- Felicia Hardy (Main story and flashback)
- Mary Jane Watson (Only in flashback)
- Aunt May Parker
- Dr. Curt Connors
- Michael Morbius (First appearance)
- Debra Whitman
- Flash Thompson
- J. Jonah Jameson (Voice Only)
- Wilson Fisk/Kingpin
- Silvermane (First appearance)
- Owl (Cameo) (First appearance)
- Hammerhead (Cameo) (First appearance)
- Alistair Smythe
- Insidious Six (First appearance)
- Professor Farley Stillwell (Only in flashback)
- Spider-Man's Web-Shooters
- Shocker's Vibro-Shock Gauntlets
- Doctor Octopus' Tentacles
- Spider-Man robot (Only appearance)
- Neogenic Recombinator (Only in flashback)
- Bruce the Gargoyle
The Kingpin has a meeting with his fellow crime lords, where it appears that they are having a profitable year. However, Silvermane is less than happy with the success, with Spider-Man still a threat to their organisations. Silvermane has the Kingpin restrained, but he is able to break free. Kingpin then announces that he has begun an operation which will eliminate Spider-Man.
In prison, the Chameleon receives a message from Alistair Smythe telling him that the Kingpin is breaking him out of prison, and Smythe orders him to release Mac Gargan, Quentin Beck and Shocker, who then use their abilities to release Rhino and Doctor Octopus from their cells. The six fugitives then escape in a helicopter before they meet the Kingpin, who tells them that he has banded them together as the Insidious Six. They agree to work together to destroy Spider-Man, and although reluctant at first Doctor Octopus agrees to the plan.
Meanwhile, Spider-Man notices that he seems to be losing his powers, as he slips while wallcrawling and nearly suffers from a lethal fall. He goes home, where he sleeps for a whole day and night, before waking up to find he is late for science class. Peter arrives late for class, where a contest is being held over several weeks for the chance to become Dr. Curt Connors's research assistant. After class, Felicia Hardy asks Peter out on a date but they are interrupted by Flash Thompson, who tells Peter that there are several of Spider-Man's villains causing trouble around the city. As Spider-Man confronts the Insidious Six, he is almost defeated by them having partially lost his powers, but manages to escape on a train.
Spider-Man visits Dr. Connors, hoping to find out why he is losing his powers. Connors tells him that his DNA structure is continuing to change, as a result of the initial radiated spider bite which had granted him his spider powers. This means that Peter is late for his fate with Felicia, who instead goes off with Michael Morbius after he saves her from being mugged.
The Insidious Six come up with a plan to capture Spider-Man, deceiving his Aunt May by telling her that Peter collapsed on the street and had been taken to "Dr. Octavius's clinic". Peter returns home to find a note demanding that he send Spider-Man to them if he is to ensure Aunt May's safety. As Spider-Man, he goes to the warehouse, which has been made to appear as a clinic by Mysterio. There, he is confronted by the Insidious Six.