We have established a hybridoma producing monoclonal antibody (MAb) against a linear epitope of glycoprotein (G protein) of the RC-HL strain of rabies virus. This MAbl5-13 showed almost the same neutralizing activity to all of five rabies fixed strains, including RC-HL, and reacted to the denatured G protein in western blot analysis. To characterize and map this linear epitope, an antigenic variant NR15-13 was selected from RC-HL strain in the presence of neutralizing MAbl5-13. The variant reacted with MAbl5-13 in an immunofluorescent antibody test but was not neutralized by the antibody and the antibody did not bind to the variant G protein in a Western blot analysis. The variant NR15-13 had an amino acid substitution at position 251 of the G protein, where tryptophan of the parental RC-HL strain was replaced by arginine. Site-directed mutagenesis analysis using the expression system in simian COS7 cells revealed that a single amino acid substitution at 251-tryptophan by arginine on the G protein of the parental RC-HL strain abolished the antigenicity of the epitope for MAbl5-13 in western blot analysis, and the replacement of 251-arginine by tryptophan recovered the activity. These results strongly suggest that tryptophan at position 251 on the G protein is essential for construction of the linear epitope against MAbl5-13.