Half of the protein S in plasma is present as a complex with a C4b-binding protein (C4bp), a complement component (Mr 570,000). In this study, the protein S-binding site on C4bp was examined by using monoclonal anti-C4bp-IgGs. C4bp was cleaved by chymotryptic digestion into seven NH2-terminal arm fragments (Mr 48,000) and a COOH-terminal core fragment (Mr 160,000). The COOH-terminal fragment inhibited the cofactor activity of protein S and its binding to C4bp in a dose-dependent manner. A monoclonal anti-C4bp-IgG (MFbp16), which binds to the COOH-terminal fragment, inhibited the binding of protein S to C4bp. The chymotryptic digest of the reduced and carboxymethylated COOH-terminal fragment was subjected to MFbp16-Sepharose 4B column affinity chromatography, and a peptide of Mr 2,500 was obtained. Protein S bound to the Mr 2,500 peptide, and this binding was inhibited by C4bp in a dose-dependent manner. The sequence of this peptide corresponded to Ser447-Tyr467 near the COOH terminus of the C4bp subunit. MFbp16, which bound to Mr 570,000 C4bp (C4bp-high), did not bind to Mr 510,000 C4bp (C4bp-low) in human plasma that does not form a complex with protein S. This suggests that C4bp-low lacks the protein S-binding site present in the COOH-terminal region of C4bp-high. Since C4bp-low also dissociates into identical subunits when reduced, the interchain disulfide bond region that links the seven subunits of C4bp appears to be closer to the NH2-terminal end than the protein S-binding site.