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Ultimate Spider-Man #13
There was already a considerable amount of buzz around Ultimate Spider-Man by the time issue #13 came out. Brian Michael Bendis had established himself as a major force at Marvel Comics, and Mark Bagley had his career completely rejuvinated - and yet they both managed to outdo themselves with the critically acclaimed thirteenth issue, in which Peter Parker reveals his secret identity to Mary Jane Watson.
While I mentioned Bagley, this issue was almost completely about the writing (if I recall correctly, a great deal of the panels were photostats) of Bendis, as he explored the reaction of Mary Jane as well as the reaction of Peter to her reaction - all the while, clearly establishing that Ultimate Spider-Man was departing from the style of stories that Amazing Spider-Man was telling dramatically.
The issue, which was almost entirely dialogue, was later revisited by Bendis on the title a few times. In fact, I would say that most of the most notable issues of this popular series were the ones where Bendis is given room to just create a sort of two-person mini-play. The recent issue with the Shocker and Spider-Man (#122?) followed in the footsteps of this issue, with its tense dialogue between two characters.
The first appearance of Mysterio in Amazing Spider-Man #13 is certainly important, but I do not think that issue stands out as much as this issue does - when you think of Amazing Spider-Man issues, #13 would not crack the top twenty. If you think of Ultimate Spider-Man issues, #13 is at or right next to the top of the list, and the book maintains a nice cultural resonance.