Inhibitory antibodies directed against factor VIII develop in a substantial number of patients with hemophilia A as a consequence of factor VIII replacement therapy. These antibodies usually recognize discrete epitopes within the A2 and/or the C2 domains of factor VIII. Here, we have characterized the antibodies present in the plasma of a patient affected by severe hemophilia A. The antibodies reacted readily with the metabolically labeled factor VIII light chain and fragments thereof when analyzed by immunoprecipitation. The inhibitory activity could be neutralized by the complete light chain, whereas only slight neutralization occurred with a fragment comprising the isolated C2 domain. Binding of the majority of antibodies to in vitro synthesized factor VIII fragments was dependent on the presence of amino acid residues Gln1778-Met1823, a region known to contain a factor IXa binding site. Functional characterization showed that purified IgG from the patient's serum inhibited binding of factor IXa to immobilized factor VIII light chain in a dose-dependent manner. These data indicate that human alloantibodies may inhibit factor VIII activity by interfering with factor IXa-factor VIIIa complex assembly.