- Spider-Man (Peter Parker) (First appearance) (Origin revealed) (Main story and flashback) (Joins S.H.I.E.L.D.)
- S.H.I.E.L.D. (First appearance)
- Nick Fury (First appearance)
- Mary Jane Watson (First appearance) (Main story and flashback)
- Harry Osborn (First appearance) (Main story and flashback)
- Flash Thompson (First appearance) (Main story and flashback)
- Stan the Janitor (First appearance)
- May Parker (First appearance) (Main story and flashback)
- Hydra (First appearance) (Cameo)
- Burglar (First appearance) (Only in flashback)
- Frightful Four (First appearance)
- Oscorp (First appearance)
- Daily Bugle Communication (First appearance)
- J. J. Jameson (First appearance) (Appears on a computer screen, TV, or hologram only)
- The Thing (Ben Grimm) (First appearance)
- Avengers (First appearance) (Vision)
- Ben Parker (First appearance) (Only in flashback)
- Irving Forbush (Mentioned)
- Genetically-Modified Spider (First appearance) (Only in flashback)
- NYPD officer (First appearance)
- Midtown High Principal
- Earth-12041 (First appearance)
- Spider-Man's Web-Shooters (First appearance)
- S.H.I.E.L.D. upgraded Spider-Man's Web-Shooters (First appearance)
- Doctor Octopus' Tentacles (First appearance)
- Sonic Disruptor
- Captain America's Shield (First appearance) (Peter Parker's imagination)
- S.H.I.E.L.D. Hellicarrier (First appearance)
New York, at the Times Square, J. J. Jameson from Daily Bugle Communications is giving a report asking for the apprehension of the vigilante known as Spider-Man. Spider-Man, swings across the city until he encounters with a robbery, he saves a police officer from crashing with his car to a shop by using a web. The officer thanks Spider-Man as he jumps to the scene, where he meets the Trapster, (breaking the fourth wall) Spider-Man explains his previous three encounters with this criminal, who is more a pilferer than a super-villain. When Trapster tries to take Spider-Man down using his special glue pistol, Spider-Man punches him to a car. The villain takes two granades and trows them to the hero, he manages to avoid one, while other explodes, letting him glued to a wall. When the Trapster is about to shoot the immobilized hero, Spider-Man webs his gun's tip, causing a pressure increase and a explosion of Trapster's equipment, letting the villain totally glued, and out of combat.
As soon as the battle ends, Spidey sees S.H.I.E.L.D. Helicarrier above and Nick Fury approaches our hero from the back. Fury scolds Spider-Man for the collateral damage from the battle, saying that Captain America would have made it in just three secons, but that even Iron Man had a learining curve. Spider-Man, disinterested, is leaving the scene when Fury tells him he knows he's Peter Parker, and Spider-Man remembers his childhood, his his uncles, when he got his powers and when Uncle Ben died. "'With great power comes great responsability'. Good words from a good man" says Fury, and Spider-Man replies that Uncle Ben was the best, and it's been a year since his death, and the start of Peter's career. When Fury tells Spidey he wants him to become "the Ultimate Spider-Man", Peter asks "How ultimate is 'Ultimate'?", which Fury replies by knocking out a fugitive Trapster without even looking, by making a pole hit the villain with only one shot of his gun, "That ultimate". When Spider-Man swings away, another person was watching him trough a camera, Norman Osborn, who wants to use Spider-Man to make an army of super-soldiers, with the help of his partner, Doctor Octavius, "we will being phase two".
At Midtown High, Peter meets Mary Jane Watson, one of his best friends since they were kids. Mary Jane doesn't know Peter's secret, as he lies her when she asks him why was he late. Also MJ informs Peter she wants to work for Jameson, in her future journalism career. Harry Osborn approaches and Peter remembers when he first met him, and his father. Later, Flash Thompson, Peter's all life bully, appears and puts him in a locker, Peter is soon released by Stan the janitor.
While at the cafeteria, Peter's spider-sense tingles, and he saves MJ and Harry from a wall being torn apart. The Frightful Four, Wizard, Klaw and Thundra (minus Trapster), appears and threats to destroy the school if Spider-Man doesn't appears. Wizard mentions that Trapster managed to know where Spider-Man was. After distracting the villains by starting a food fight against them, Peter changes, and finds in his arm a tracer which Trapster left him during the battle which took place hours before.
Spider-Man joins the battle, and Wizard announces to Osborn via a communicator that they found him. Meanwhile Spider-Man battles Thundra, Klaw tries to take down Mary Jane, who was using her camera to record the battle. Harry saves her, but is hit by Klaw's sonic wave. Spider-Man kicks Wizard out of the cafeteria to the school's corridor, there, Flash appears telling Spider-Man he was his biggest fan and wanted to help him. To get "revenge" from what Thompson made to him as Peter, Spider-Man asks Flash to hide in a locker, to attack by surprise the villains. Spider-Man does the same in the ventilation systems, but the "Frightful Three" escapes when they hear sirens.
Back as Peter, he witness when Harry is taken to a hospital. Back at Aunt May's house, Peter tells her the situation that happened. While reflecting about his life, Peter relizes he has to take Fury's offer. At night, Spider-Man arrives to the Helicarrier, where after destroying most of the security systems, he's totally pointed by guns, Fury appears and deactivates them. Peter says "Ultimate Spider-Man reporting for duty... sir!" and Fury replies "Welcome to S.H.I.E.L.D., Spider-Man. Hope you survive the experience".
- This issue was adapted in Ultimate Spider-Man: Premiere Comic, which came out 2 weeks before the premiere of the show as a preview.
- The Iron Man's wacky flight scene is reminiscent of Tony Stark's first flight tests in the Earth-199999's Iron Man movie.
- Peter complaining that working for Jameson would be a nightmare is possibly an ironic nod to the character's initial history in the comics working for him.
- Klaw remarks that Midtown High is full of would-be heroes. This statement proves true as its student body would produce Agent Venom, Iron Spider, Patrioteer and of course, Spider-Man himself.