The development of an immune response towards factor VIII (fVIII) remains a major complication for hemophilia A patients receiving fVIII infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody. Molecular analysis of the specific immune response is therefore required. To this end, we have characterized an fVIII-specific human IgG4kappa monoclonal antibody (BO2C11) produced by a cell line derived from the memory B-cell repertoire of a hemophilia A patient with inhibitor. BO2C11 recognizes the C2 domain of fVIII and inhibits its binding to both von Willebrand factor (vWF) and phospholipids. It completely inhibits the procoagulant activity of native and activated fVIII, with a specific activity of approximately 7,000 Bethesda units/mg. vWF reduces the rate of fVIII inactivation by BO2C11. The antibody-fVIII association rate constant (kass approximately 7.4 x 10(5) M-1 s-1) is eightfold lower than that for vWF-fVIII association, whereas its dissociation rate constant (kdiss < or = 1 x 10(-5) s-1) is 100-fold lower than that for the vWF-fVIII complex, which suggests that BO2C11 almost irreversibly neutralizes fVIII after its dissociation from vWF. BO2C11 is the first human monoclonal anti-fVIII IgG antibody that has been isolated and allows the study of fVIII inactivation at the molecular level.