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Mysterio (voiced by Gregg Berger) was a supervillain who blames Spider-Man for ruining his reputation. In his first appearance, Mysterio frames Spider-Man for various crimes, but his plan is exposed by Spider-Man and Detective Terri Lee, and he is jailed. Later, he becomes a member of the Insidious Six in the episodes"The Insidious Six" and "Battle of the Insidious Six", but the entire team fails and disbands to avoid being arrested. In Mysterio's final appearance in the series, "The Haunting of Mary Jane", his ex, Miranda Wilson, creates a studio in secret and kidnaps the clone of Mary Jane Watson and Spider-Man teams up with Mysterio, battling robot versions of villains Spider-Man fought in the past in a deathtrap Mysterio had designed to one day lure Spider-Man to and kill him. Spider-Man discovers that Mysterio was in love with a woman named Miranda Wilson, a former actress who was disfigured and planned the entire kidnapping to swap bodies with the similar-looking Mary Jane. The studio exploded, Spider-Man saved the Mary Jane, and Mysterio dies in the explosion, staying with Miranda to the end. In the five-part "Six Forgotten Warriors" storyline, in which the Kingpin hires the Insidious Six again, the Vulture replaces the late Mysterio.

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