The properdin-binding site in the human third complement component (C3) was mapped by using isolated C3b, C3c, alpha- and beta-chains of C3 and C3 polypeptide fragments and an enzyme-linked-immunosorbent-assay procedure. The C3 chains and the polypeptide fragments were purified to homogeneity by preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The alpha-chain polypeptides included a 68 kDa and a 43 kDa polypeptide, which were generated by cleavage of C3b with factors I and H, and a 40 kDa, 33 kDa (C3d) and 27 kDa polypeptide, which were generated by cleavage of C3b with porcine elastase. It was shown that properdin binds to C3b, C3c, alpha-chain, and to the 43 kDa (factor-I + H-derived), as well as to 40 kDa (elastase-derived) alpha-chain fragment, but not to the beta-chain 68 kDa, 33 kDa (C3d) and 27 kDa alpha-chain fragments. Thus the binding site for properdin resides on the 40-43 kDa C-terminal alpha-chain fragment of C3.