Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the membrane attack complex (MAC). C8 is an oligomeric protein composed of a disulfide-linked C8α-γ heterodimer and a noncovalently associated C8β chain. C8α and C8β are homologous; both contain N- and C-terminal modules and an intervening ∼40 kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. C8β participates in at least two binding interactions. It has a high affinity binding site for C8α, which facilitates its interaction with C8α-γ. C8β also mediates incorporation of C8 into the MAC by binding to C5b-7, an intermediate in the MAC assembly pathway. Little is known about the location or properties of the respective binding sites on C8β. In this study, the MACPF domain of C8β (βMACPF) was expressed in Escherichia coli and its role in binding C8α and C5b-7 examined. Recombinant βMACPF was shown to bind C8α-γ in solution and form a noncovalent complex (βMACPF•C8α-γ) that exhibited C8 hemolytic activity. βMACPF was also capable of binding independently to erythrocytes carrying C5b-7. Subsequent addition of C8α-γ and C9 to these cells produced a hemolytically active MAC. The ability to produce a soluble, recombinant βMACPF that retains the binding functions of C8β suggests this segment of C8β is an independently folded domain. Furthermore, results indicate the principal binding sites for C8α and C5b-7 are located within this domain, and that C8β binding specificity is not determined by the N- and C-terminal modules.