- The Symbiote
- Aunt May Parker
- Mary Jane Watson
- Daily Bugle
- Captain George Stacy
- Anna Watson
- The Sinister Six (First appearance)
- The Enforcers
- The Lizard (Curt Connors) (Mentioned)
- Green Goblin (Mentioned)
- Norman Osborn (Mentioned)
- Eddie Brock
- Dr. Ashley Kafka (First appearance)
- Officer Jean DeWolff
- Officer Stan Carter
- Officers Morales & Wingard
- St. John Devereaux
- Dilbert Trilby (Appears on a computer screen, TV, or hologram only)
- Ben Parker (Photo)
- Kenny "Kong" McFarlane (Mentioned)
- Peter & Eddie's Parents (Mentioned)
Races and Species:
- Spider-Man's Webshooters
- The Symbiote Suit
- Doctor Octopus' Tentacles
- Shocker's Vibro-Shock Gauntlets
- Vulture's Wings
- Rhino Armor
- Civilian Cars and Buses
- Cop Cars
- An Ambulance
- Eddie Brock's Motorcycle
Due to good results from psychiatrists, Max Dillon is released from Ravencroft Institute, albeit with his suit still on, by Dr. Kafka. Immediately after leaving, he gives a call to Otto Octavius, now in prison on Ryker's Island, who had written to him about an evil plot while he was in confinement. They both confirm their intentions, and then Octavius signals Adrian Toomes to gather up more villains. Toomes gives lunch to an irritated and heavily confined Rhino and Sandman, giving a subtle hint of what is to come: Spider-Man is dessert.
At night, Electro arrives and causes a distraction that kills two guards and shuts off the power. Rhino and Sandman break out of their cells and then break Octavius, Toomes, and three other prisoners out of prison. Electro brings them out, and Hammerhead arrives and gives them their suits before they head for the mainland.
Meanwhile, Peter wakes up at 12:16 in the afternoon and discovers that the black costume can make its own webs. Heading downstairs, he finds that Aunt May is really tired and does not want to go to the movies with Anna Watson as she had planned earlier. Peter suggests she goes, since she really needs it. Peter later arrives at Empire State University, where he runs into Eddie. Eddie brushes him off, saying that the thing with Peter supposedly being a bystander photographer as the Lizard and the Black Cat did whatever crime they wanted officially no longer made them friends no more. He also says that Peter probably doesn't care about anyone, and needs a taste of his own medicine.
Peter feels ashamed that he lost his oldest friend, but brushes it off, realizing he has the new suit. Seeing a police chase, he follows it to where Rhino is on a rampage just outside the movie theater where Aunt May is. Spider-Man goes to fight him, when suddenly Sandman, Shocker, Vulture, Electro, and Doc Ock appear to form the Sinister Six - and attack Spider-Man.
Spider-Man gets Aunt May and Anna Watson into the safety of the theater, where May collapses. Spider-Man continues fighting, but can't lay a hit on any of them, so he bails out into the sewer. Even though all except Shocker and Doc Ock are unable to make their way in there, Spidey doesn't do anything. Meanwhile, Eddie gets a date with Mary Jane, presumably to attack Peter.
At the Daily Bugle, Jameson gets angry that Spider-Man "helped" the Sinister Six. Robbie Robertson then reveals there were no casualties, but that May Parker had a heart attack. Jameson calls Peter at home, but Peter, thinking that he is going to rant about not getting photos, ignores him. He appears oblivious to May's disappearance, and is asleep when Anna Watson calls him frantically.
The Sinister Six, with the police right outside, sit for dinner. They end up arguing, but Electro calms everyone down. Doc Ock then says they will need to draw out Spider-Man if they are to kill him. They take hostages at a bank. Spider-Man goes there, but is instantly caught. As Rhino prepares to execute Spidey, webs come out of his armpits and he escapes again into Central Park.
Eddie recklessly takes Mary Jane on the highway on a motorcycle while explaining the background between his relationship with Peter, and it is indirectly revealed that he is targeting her specifically because Peter took her to the dance. When his driving gets far too out of control, MJ leaves.
The police, including Captain Stacy, arrive as Spider-Man fights the Sinister Six in the park. Throughout the battle, Peter doesn't say a word. He defeats Rhino and Electro before defeating Vulture, Sandman, and Shocker. Doc Ock captures him and tries to impale him on Rhino, but Peter gets away from his grasp again. He defeats Doc Ock and nearly kills him, but the police tell him not to and he leaves.
Peter wakes up the next morning with no memory of the fight. Seeing it in the paper, he thinks that the suit, which he now calls a symbiote, controlled him to fight. He angrily brushes it off and heads back in when MJ shows up and tells him that Aunt May had a heart attack and is now in the hospital.
- Production Code 111