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In Spider-Man: The Animated Series, Mary Jane Watson (voiced by Sara Ballantine) first meets a 19-year-old Peter Parker in "The Return of the Spider-Slayers" (Season 1, episode 4), where May Parker set Peter up on a blind date with her. At the time, Peter's main love interest was Felicia Hardy and he did not look forward to meeting Mary Jane, but is left speechless when he meets her. She is last seen in the season three finale "Turning Point," in which the Green Goblin discovers Spider-Man's true identity. In a nod to "The Night Gwen Stacy Died", he takes her to the George Washington Bridge. Spider-Man tries to save her, but she falls into a dimensional portal created by his stolen time dilation accelerator. In season four, a clone of Mary Jane, created by Miles Warren, appears, and after the death of Mysterio at the end of Season 4, Episode 9, Spider-Man reveals his secret identity to her. They subsequently marry in the Season 5 premiere, when she is revealed to be a clone, one that possesses the same powers as Hydro-Man, but by the end of the episode, her body disintegrates as a result of imperfections in the cloning process. In the series finale, Spider-Man's mystic advisor, Madame Web, promises they will find the real Mary Jane.

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