In a previous study, we prepared a monoclonal antibody (MoAb) to coagulation factor IX (FIX), designated 65–10, which interfered with the activation of FIX by the activated factor XI/Ca 2+ and neutralized the prolonged ox brain prothrombin time of hemophilia B M [11,12]. The location of the epitope on the FIX for 65–10 MoAb is 168 Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val 182 [21]. In this paper, we studied in more detail an epitope on FIX using the systematic substitution of different amino acids at each residue of the epitope peptides and the influence of the epitope peptide on the prolonged ox brain prothrombin time of the hemophilia B M plasma of 65–10 MoAb. In the replacement set of amino acids, peptides showing low or no reactivity to 65–10 were 175Phe → Asp, Glu, Gly, Lys, Arg, Thr, Val, 176Asn → Asp, Glu, Phe, Ile, Lys, Leu, Pro, Val, Tyr, 177Asp → Cys, Glu, Phe, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, and 178 Phe → Pro. These results imply that a hydrophobic molecule of 175 Phe, a hydrophilic molecule of 176Asn, and a negative charge molecule of 177Asp were important to the epitope. The 65–10 MoAb antibody neutralized the prolonged ox brain prothrombin time of hemophilia B M Nagoya 2 ( 180Arg →Trp) and Kashihara ( 181Val → Phe) as well as B M Kiryu ( 313Val → Asp) and Niigata ( 390Ala → Val). This reaction was inhibited by preincubation with a 168 Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val 182 peptide conjugated with bovine serum albumin (BSA). 65–10 MoAb that has been useful in detailing epitopes will be useful for qualitative analysis of hemophilia B M.