Perfringolysin O (PFO), a member of the cholesterol-dependent cytolysin family of pore-forming toxins, forms large oligomeric complexes comprising up to 50 monomers. In the present study, a disulfide bridge was introduced between cysteine-substituted serine 190 of transmembrane hairpin 1 (TMH1) and cysteine-substituted glycine 57 of domain 2 of PFO. The resulting disulfide-trapped mutant (PFOC190-C57) was devoid of hemolytic activity and could not insert either of its transmembrane β-hairpins (TMHs) into the membrane unless the disulfide was reduced. Both the size of the oligomer formed on the membrane and its rate of formation were unaffected by the oxidation state of the Cys190-Cys57 disulfide bond; thus, the disulfide-trapped PFO was assembled into a prepore complex on the membrane. The conversion of this prepore to the pore complex was achieved by reducing the C1900-C57 disulfide bond. PFOC190-C57 that was allowed to form the prepore prior to the reduction of the disulfide exhibited a dramatic increase in the rate of PFO-dependent hemolysis and the membrane insertion of its TMHs when compared with toxin that had the disulfide reduced prior mixing the toxin with membranes. Therefore, the rate-limiting step in pore formation is prepore assembly, not TMH insertion. These data demonstrate that the prepore is a legitimate intermediate during the insertion of the large transmembrane β-sheet of the PFO oligomer. Finally, the PFO TMHs do not appear to insert independently, but instead their insertion is coupled.