Protection of host cells from lysis by the C5b-9 cytolytic complex is provided by a membrane-associated protein (CD59) that interacts with homologous C8 and C9 during C5b-9 assembly. This interaction restricts normal formation and function of the complex, thereby protecting cells from attack by homologous C. In this study, rabbit C8 was purified and characterized and used to investigate the role of the individual C8 subunits in homologous restriction and the basis for species selectivity by human CD59. Exchanging the disulfide-linked C8 alpha-gamma dimer in human C8 with one from rabbit was found to be sufficient to overcome restriction by human Es. Activity of the hybrid C8 and rabbit C8 toward these target cells was equivalent, thus establishing that restriction is not dependent on the species of C8 beta. Because C8 gamma was previously shown to have no role in restriction, these results suggest that within C8, structural determinant(s) recognized by CD59 reside solely on C8 alpha. Sequences determined from full-length cDNA clones for rabbit C8 alpha, C8 beta, and C8 gamma support this conclusion. All three subunits are strikingly similar to human with regard to length, m.w., N- and C-terminal residues, conserved cysteines, and overall sequence. However, when sequences are compared under high stringency, a single segment of extended sequence dissimilarity is detected between the two species of C8 alpha. Within human C8 alpha, this 37-residue segment resides near the putative membrane-interacting region and may constitute a human CD59 recognition site.