Hemophilia B Kashihara is a severe hemorrhagic disorder in which the factor IX antigen is present in normal amounts but factor IX biological activity is markedly reduced. In addition, purified factor IX Kashihara is not activated by purified factor XIa in the presence of calcium ions. Amino acid sequence analysis of one of the tryptic peptides isolated from factor IX Kashihara indicated that Val-182 (equivalent to Val-17 in the chymotrypsin numbering system) had been replaced by Phe. No substitution was found in the members of the catalytic triad His-221, Asp-269, and Ser-365 of factor IX Kashihara. The Val-to-Phe replacement found in factor IX Kashihara appears to sterically hinder the cleavage of Arg 180-Val 181 by factor XIa required for the activation of this zymogen.