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The game's main protagonist is Leon S. Kennedy, one of the few survivors of the Raccoon City incident, who was recruited and trained by the US Government to become a special agent after they learned of his actions in Raccoon City. Now an agent for a special security agency, Leon is sent on a mission to rescue Ashley Graham, the President's daughter, who has been kidnapped by a mysterious cult organization. Leon travels to an undisclosed village in Spain where he encounters a horde of unruly villagers who pledge their lives to Los Illuminados, the cult that perpetuated Ashley’s kidnapping. He meets Luis Sera, a former Los Illuminados researcher, who aids Leon on his mission. By examining Sera’s notes, Leon discovers that Los Illuminados gained control of their subjects by implanting a mind controlling parasite known as "Las Plagas" into their bodies. Unfortunately, Leon and Luis are captured, during which time Leon is injected with the parasite. After escaping and meeting a mysterious man known as the Merchant, who supplies Leon with ordanance and weaponry, Leon returns to his objective. He travels across a lake where he faces large creature "Del Lago". Leon defeats the creature, but is severely fatigued, and collapses in a cabin. After regaining consciousness, he obtains a key to open the church where Ashley is being held. On the way back Leon fights with gigantic "El Gigante". While escaping with Ashley, he meets the mastermind of Los Illuminados: Osmund Saddler. He tells them about the Plagas growing in their bodies. The two escape from Saddler, and make their way to the extraction point. Meeting up with Luis once more in a cabin, they are ambushed by a seemingly never-ending crowd of Ganados. When the attack ends, Leon and Luis separate. However, Leon finds out that the helicopter has been shot down. After traveling through a canyon, where the two face another El Gigante.Traveling down a gondola, and facing village chief Bitores Mendez, Leon takes his false eye, and uses it on a retinal scan to open a door, which leads to the castle. Leon and Ashley believe they can take refuge in it until another chopper arrives. They were wrong.
After meeting yet another of Saddler's acolytes, the castle's castellan, Ramon Salazar, who jacks Leon's communication with Washington, Leon and Ashley travel to the center of the castle. But Ashley starts coughing blood, which distresses her, and she runs off, and is captured. While searching for Ashley, Leon meets Ada Wong, who is there for the sample of the "Master Plaga", but she doesn't tell Leon about it. Then Leon fights his way through the underground sewer system, despite assaults by Saddler's mutated "Novistadors". When Leon finds Ashley and is about to rescue her, Luis runs in with drugs to suppress the growth of the parasite, and a sample of Master Plaga for the U.S. government, when he is killed by Saddler. Saddler retrieves the sample and leaves. Leon rescues Ashley, but vows to avenge Luis's death. While Leon can't get to Ashley, she must find a way to him. She is, however, caught again and sent to the military research island, forcing Leon to travel to there too with Ada. After numerous altercations with Saddler's fiends, Leon is able to successfully rescue Ashley, and run from Saddler with Ada's assistance.
After an intense fight with Saddler, he finds the Sample but Ada forces him to give it to her. She then escapes from the complex on a helicopter, leaving Leon and Ashley to escape via jet-ski. Although Leon and Ashley manage to escape from the island, the sample's whereabouts and fate remain uncertain.