Following the theft of a deadly virus, code named Substance 33, in Turkey, Sydney is forced to partner on a mission with new APO agent Thomas Grace where they and Marshall travel to Prauge to look for the stolen virus container where their paths cross with Rachel Gibson, a spy suspected of being involved in activities against the U.S., has something shockingly in common with Sydney that she's working for a black ops group called The Shed, that is run by Gordon Dean and his murderous associate Kelly Peyton. Sydney and Tom must persuade Rachel to infiltrate The Shed to download Dean's computer files to learn where Substance 33 is being hidden. Meanwhile, Dixon must accompany Sloane, who's given a 48-hour pass from prison to acquire a possible cure for his still-comatose daughter, Nadia, from a scientist in Russia.

While Sloane awaits the outcome of his sentencing for treason, a mysterious associate of Gordon Dean approaches him for a favor in exchange for persuading the judges to rule in favor of acquittal. Meanwhile, Sydney and Tom must try to convince Rachel to help them catch the fugitive Gordon Dean who moves his operations to more secure locations. Sydney and Rachel return to the ruins of the Shed's offices in Prague for Rachel to reconstruction Dean's computer server so Tom and Dixon in the Cayman Islands can empty out Dean's bank accounts. Although they are successful, Dean discovers that Rachel is still alive which puts both her and Sydney in grave danger when they are trapped in a car that's hanging by an electromagnet controlled by Dean and his lethal associate Peyton who threaten to drop the hovering car unless Rachel's location is revealed.

After Sydney and the APO team infiltrates a formal party at the Chinese embassy in India to locate a fugitive arms dealer, named Janos Vak, Rachel is chosen to begin very first solo mission by infiltrating an off-shore oil platform, with com-links from Sydney whose pregnancy now limits her role in missions, to steal some computer software from Janos to gain the location of a new weapon. Meanwhile, Sloan, now back at APO, is in frequent contact with the elusive Gordon Dean who wants him to keep close tabs on APO, leading to Dean sending his lethal right-hand assassin, Kelly Peyton, to recover the computer software before APO does leading to a hand-to-hand fight between Peyton and the field-inexperienced Rachel.

Sloane decides to betray Dean when he learns that Nadia's condition will probably not improve. They meet at a race track in Dubai to exchange a network access card which Sydney and Renee stole in Rome, leading to Dean finally being captured. The APO interrogates Dean for information on Prophet Five. Yet another mysterious stranger asks Sloane to eliminate Dean. Meanwhile, Nadia comes out of her coma for a short time after a nameless nurse leaves her room. Under orders from his new Prophet Five contacts, Sloane kills Dean during a routine reboot of the APO's surveillance network, and APO misses its chance to hack the network, but learns Prophet Five is linked to intelligence agencies around the world. At the end, control of Dean's terrorist cell is given to Kelly Peyton.

When Kelly Peyton steals some software from a CIA warehouse concerning the late Michael Vaughn, Sydney is soon abducted by Prophet Five agents where she is subjected to a hypnotic state making her flash back to her happier times with Vaughn in order to learn where a supposedly powerful device, called The Horizon, is located. Meanwhile, Jack must team up with Renée to locate Sydney when they captured and torture Aldo Desantis, while the mastermind behind Prophet Five is finally revealed... and why the person has a strong interest in Sydney's unborn daughter.

After escaping from Peyton and her henchmen, and learning she's on a freighter at sea, Sydney goes to the communications room and sends a message to APO routed through the CIA to her whereabouts. Unbeknownst to her, the message is intercepted by a mole at the CIA working for Prophet Five, although APO was able to obtain a piece of the transmission. Wanting to retrieve Sydney, the APO team, Rachel, Tom, Dixon, and Marshall attempt to break into a secured server room located at Langley with the goal of obtaining the remaining part of the transmission. Problems arise, however, when APO is forced to take the hard drive, which triggers an alarm. They manage to escape the situation thanks to former APO agent Eric Weiss who aids them in sneaking past the guards. Although the transmission on the hard drive is corrupted, APO is able to learn who corrupted it. Jack sets a meeting up with the division heads of the CIA and is quickly able to identify the mole. Meanwhile, Tom is revealed to have another agenda when he obtains information about a certain Allan Korman, who was a former German assassin now in a witness relocation program, and who murdered Tom's wife many years earlier.

Now nine months pregnant and on maternity leave, Sydney is surprised when her mother, Irina Derevko, shows up at her front door pretending to be a victim pursued by Prophet Five to get information on the whereabouts of 'The Horizon'. Jack, Sydney and Irina travel to Vancouver to an high-rise office building to steal the Horizon, only to get trapped on the upper floors when Peyton and hit team show up, forcing Jack to do battle with them alone as Sydney finally begins to go into labor with Irina as her labor coach. Elsewhere, while Dixon meets with Renee in Paris for information about The Horizon, back at APO, Rachel, Marshall and Tom agree to cover for Sloan when CIA Director Arthur Devilin arrives to monitor the APO records in the wake of the discovery of Prophet Five mole within the CIA.

One month later, with Sydney on maternity leave, she gets forced back into the field when her old enemy, Anna Espinosa, returns after she's secretly sprung from prison by Peyton who gives her a job offer to work for Prophet Five whose leaders have secretly contact with Sloan to get him to set up Sydney in exchange for getting the cure for Nadia. Anna decides that to get at Sydney is that she needs to go through her friends, leading to Anna to abduct Will Tippen. Sydney, along with APO, is forced to try to find Anna and save Will before he's killed unaware that Prophet Five has bigger plans for Syd, to take a sample of her DNA.

While Sydney and the rest of APO try to learn more about Prophet Five and what their plans for Sydney are, Sloane finally acquires a bottle of serum from Prophet Five as a cure for the comatose Nadia. But Sloane becomes distraught when he learns that for it to work, he must stop Nadia's heart for a full 30 seconds. When the proceeder is successful, Jack becomes suspicious about Sloane's possible involvement with Prophet Five, leading him and Nadia to investigate, while the rest of the APO team continues searching for Anna Espinosa, unaware that she has been duplicated by Project Helix to look like Sydney. Meanwhile, Tom travels to France to meet with Renee to look for a fugitive art dealer, named Moritz, whom has the stolen Rimbalid Page 47 artifact, and learn that it's a fake for the real one has with Sloan all this time. Also, Sydney offers Renee a job at APO, only to have fatal confrontation with Anna/Sydney, while Nadia has her own confrontation with her father over his renewed obsession with Rimbaldi.

Visions trouble Sloane while he works for Prophet Five in deciphering the Rimbaldi Page 47 artifact. Meanwhile, a computer chip is found in the dead Renée's body leading Sydney to try to make contact with Vaughn.

Sydney must remain undercover and work with Sark to track down Rambaldi's final clue located in a prison in Italy before Sloan, Peyton, and the rest of Prophet Five finds it.

Sloane takes Rachel and Marshall hostage to force them to help him find the Rambaldi clue that he is missing. Sydney, Vaughn and APO try to save them by having Marshall's wife, Carrie, to help out, while Sloan plots with Sark and Peyton to take over Prophet Five for themselves.

In the aftermath of the destruction of APO, Sydney, Jack and Vaughn must find a way to stop Irina Derevko and Arvin Sloane from fulfilling the Rambaldi endgame of Armageddon.