Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex (MAC). It is an oligomeric protein composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. C8alpha and C8beta are homologous; both contain an N-terminal thrombospondin type 1 (TSP1) module, a low-density lipoprotein receptor class A (LDLRA) module, an extended central segment referred to as the membrane attack/perforin (MACPF) domain, an epidermal growth factor (EGF) module, and a second TSP1 module at the C-terminus. In this study, the segment of C8beta that confers binding specificity toward C8alpha-gamma was identified using recombinant C8beta constructs in which the N- and/or C-terminal modules were deleted or exchanged with those from C8alpha. Constructs were tested for their ability to bind C8alpha-gamma in solution and express C8 hemolytic activity. Binding to C8alpha-gamma was found to be dependent on the TSP1 + LDLRA + MACPF segment of C8beta. Within this segment, the TSP1 module and MACPF domain are principally involved and act cooperatively to mediate binding. Results from activity assays suggest that residues within this segment also mediate binding and incorporation of C8 into the MAC.