We have used immunoblotting to localize major epitopes for 86 FVIII inhibitor alloantibodies and autoantibodies to the amino- terminal half of the 44 kDa thrombin fragment of the FVIII heavy chain and the carboxy-terminal one fourth of the 72 kDa FVIII light chain. Two of these inhibitors, which were reactive with 44 kDa thrombin fragment, were also strongly reactive with the 54 kDa thrombin fragment. We have further localized a different epitope(s) for each of these two inhibitors by the ability of three FVIII synthetic peptides from the 54 kDa thrombin fragment to partially neutralize their anti-FVIII activity. We have also used immunoblotting to analyze FVIII inhibitor immunoglobulin class and subclass content and to detect changes in FVIII epitope specificity during the course of an inhibitor. Further studies proposed in this application include: epitope mapping of FVIII inhibitors using proteolytic or recombinant DNA produced fragments of FVIII and antibodies of defined FVIII epitope specificity; epitope mapping of FVIII inhibitors using competitive immunoassays between FVIII inhibitors and heterologous antibodies of defined FVIII epitope specificity; and definition of FVIII inhibitor epitopes using synthetic peptides of FVIII. To achieve these aims we have developed and characterized an extensive collection of immunologic reagents, including 10 monoclonal anti-FVIII antibodies whose epitopes are known to within 6-40 amino acid residues of the FVIII sequence; 104 FVIII synthetic peptides; 47 rabbit antibodies to FVIII synthetic peptides 15 amino acid residues in length, which also recognize FVIII; and a collection of 86 FVIII inhibitor plasma samples. The proposed work is directed toward the long term goal of developing new therapeutic products for FVIII inhibitors.