We have recently demonstrated that protein S impairs the intrinsic tenase complex, independent of activated protein C, in competitive interactions between the A2 and A3 domains of factor VIIIa and factor IXa. In the present study, we have identified a protein S-interactive site in the A2 domain of factor VIIIa. Anti-A2 monoclonal antibody recognising a factor IXa-functional region (residues 484-509) on A2, and synthetic peptide inhibited the A2 binding to protein S by approximately 60% and approximately 70%, respectively, in solid-phase binding assays. The 484-509 peptide directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484-509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide). The cross-linked adduct was consistent with 1:1 stoichiometry of reactants. Cross-linking formation was blocked by addition of the 484-497 peptide, but not by the 498-509 peptide. Furthermore, N-terminal sequence analysis of the 484-509 peptide-protein S adduct showed that three sequential residues (S488, R489, and R490) in A2 were not identified, suggesting that these residues participate in cross-link formation. Mutant A2 molecules where these residues were converted to alanine were evaluated for the binding of protein S. The S488A, R489A, and R490A mutants demonstrated approximately four-fold lower affinity than wild-type A2. These results indicate that the 484-509 region in the A2 domain of factor VIIIa, in particular sequential residues at positions 488-490, contributes to a unique protein S-interactive site.