Four months after Vaughn's murder by the traitorous Gordon Dean and his associate Ivan Curtis, a grief-stricken Sydney teams up with Renée Rienne, a French freelance assassin and fugitive on the CIA's most wanted list who knew Vaughn from their past association with Prophet Five, to ask for leads in order to track down the killers before they can strike again. Meanwhile, Jack enlists a reluctant new agent, Thomas Grace, as Vaughn's replacement at APO, and Weiss struggles to decide whether or not to accept a promotion that would force him to move to Washington D.C. and leave his colleagues at APO. During this time, Sydney, Dixon and Weiss travel to Amsterdam to track down Curtis who is in the process of buying a potential weapon.

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.