Courtesy of her Wikipedia entry:
In the main continuity, The Amazing Spider-Man vol. 2 #35, #38:
May finally learned about her nephew's secret life when she walked into his room after he had sustained a serious beating from the villain Morlun, the two later talking about May's discovery as May came to accept her nephew's real life.
The One More Day storyline reversed this, much as it did with everything good in the universe.
In the Ultimate continuity, starting with Ultimate Spider-Man #99:
Peter reveals his secret identity to Aunt May after he finds Gwen Stacy's clone at her old house, resulting in May evicting Peter from her home, as she despises Spider-Man. .... She is rescued from dying by Sue Storm of the Fantastic Four, reconciles with Peter and accepts his life as Spider-Man, though she is not at all fond of his costume. Peter later explains to May why he became Spider-Man, and May tells him that Ben would be proud of him for doing so.
This one stuck with the end of the comic book line meaning they couldn't "fix" it.