In previous studies have shown that the interaction between factor IXa and VIII involves the light chain of factor VIII and that this interaction inhibited by the monoclonal antibody CLB-CAg A against the factor VIII region Gln1778-Asp1840 (Lenting, P.J., Donath, M.J.S.H., van Mourik, J.A., and Mertens, K. (1994) J. Biol. Chem. 269, 7150-7155). Employing distinct recombinant factor VIII fragments, we now have localized the epitope of this antibody more precisely between the A3 domain residues Glu1801 and Met1823. Hydropathy analysis indicated that this region is part of a major hydrophilic exosite within the A3 domain. The interaction of factor IXa with this exosite was studied by employing overlapping synthetic peptides encompassing the factor VII region Tyr1786-Ala1834. Factor IXa binding was found to be particularly efficient to peptide corresponding to the factor VIII sequences Lys1804-Lys1818 and Glu1811-Gln1820. The same peptides proved effective in binding antibody CLB-CAg A. Further analysis revealed that peptides Lys1804-Lys1818 and Glu1811-Gln1820 interfere with binding of factor IXa to immobilized factor VIII light chain (Ki approximately 0.2 mM and 0.3 mM, respectively). Moreover, these peptides inhibit factor X activation by factor IXa in the presence of factor VIIIa (Ki approximately 0.2 mM and 0.3 mM, respectively) but not in its absence. Equilibrium binding studies revealed that these two peptides bind to the factor IX zymogen and its activated form, factor IXa, with the same affinity (apparent Kd approximately 0.2 mM), whereas the complete factor VIII light chain displays preferential binding to factor IXa. In conclusion, our results demonstrate that peptides consisting of the factor VIII light chain residues Lys1804-Lys1818 and Glu1811-Gln1820 share a factor IXa binding site that is essential for the assembly of the factor X-activating factor IXa-factor VIIIa complex. We propose that the overlapping sequence Glu1811-Lys1818 comprises the minimal requirements for binding to activated factor IX.