We investigated the genotype distribution and allele frequency of C452T polymorphism of γ-glutamyl hydrolase (GGH) gene, which causes the decreased enzymatic activity affecting the efficacy of methotrexate (MTX), in a Japanese population. The polymerase chain reaction–restriction fragment length polymorphism assay was applied to determine the genotype of C452T polymorphism in 269 Japanese healthy individuals. The genotype distribution was as follows: C/C, 89.2% (n=240); C/T, 10.4% (n=28); T/T, 0.4% (n=1). The frequency of C and T allele was 0.944 and 0.056, respectively. The obtained genotype distribution was well agreed with those expected by Hardy–Weinberg equilibrium. The genotype distribution and allele frequency in a Japanese population were found to be similar to those of African–Americans but significantly different from Caucasians. Although the frequency of variant T allele in a Japanese population is not so high as compared to Caucasians, determination of C452T polymorphism of GGH may be useful for monitoring of efficacy and side-effects of MTX for treatment of diseases such as rheumatoid arthritis or childhood acute leukemia. To our knowledge, this is the first report about the examination of C452T polymorphism of GGH in a Japanese population.