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May's first appearance in animation was in the first season of the 1960s Spider-Man series as part of the episode "Horn of the Rhino". In the episode, she's seen trying to nurse Peter over a severe headcold and very abruptly telling J. Jonah Jameson that he was not to call Peter about photo assignments until he had recovered. May apparently made an impression on Jameson as he told Betty Brant that he did not ever want to speak to May again.

May had also made two tiny appearances in the first two episodes of the second season of the 1960s Spider-Man series. In the second season premiere where it revealed Spider-Man's origin, she and Uncle Ben made a tiny cameo about what Peter was doing after no one knew he was bitten by a radioactive spider.

In "King Pinned," she was taking medications to cure a sickness she had only it was making her feel worse. It was then revealed that it was a drug created by the Kingpin that would be fake medicine for the elderly to take that would make them feel worse to the point of death, but the plan was foiled by Spider-Man and Aunt May was saved, along with every other elderly person who took the drug.[citation needed]



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