We examined the possibility that the C2 domain, amino acid residues 2173-2332, of factor VIII (fVIII) contains a binding site for von Willebrand factor (vWf) to clarify previous data showing that some monoclonal and human inhibitor antibodies with epitopes in C2 prevent fVIII-vWf binding. We constructed a fusion protein, glutathione S-transferase-C2, which binds to immobilized vWf in a dose-dependent saturable fashion, suggesting that the fVIII C2 domain contains a binding site for vWf. This site was further localized by testing the effect of a synthetic peptide on fVIII-vWf binding. Peptide 2303-2332, consisting of a previously identified phosphatidyl-serine binding site, prevented fVIII binding to vWf, suggesting that the sites for fVIII binding to vWf or phosphatidylserine have some overlap. The effect of anti-C2 domain antibodies further supported these observations. The inhibition of fVIII binding to vWf by monoclonal antibody NMC-VIII/5 IgG or F(ab)'2 (epitope within residues 2170-2327) and by inhibitor antibody MU IgG or Fab' (epitope within residues 2248-2312) was demonstrated by a fluid-phase binding assay and enzyme-linked immunosorbent assay. Two monoclonal antibodies with epitopes within amino acid residues 2170-2218 or 2248-2285, which do not overlap the phosphatidylserine binding site, did not have any inhibitory effect. Our data suggest that the previously described antagonistic binding of vWf and phospholipid to fVIII is due to the involvement of some C2 domain amino acids in both processes.