Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338-362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338-362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338-362 would probably block this cleavage and thereby block activation of F.VIII.