Abstract The effect of 5,6,7,8-tetrahydrobiopterin (BH 4 ), an endogenous antioxidant, on γ-ray-induced micronucleus frequency was examined in RAW264.7 cells. Exogenously added BH 4 reduced the γ-ray-induced micronucleus frequency. Treatment with 1 mM sepiapterin, a biosynthetic precursor of BH 4 , also significantly reduced the micronucleus frequency induced by 3-10 Oy y-rays, by 20-35(/o at 1 8 h post-treatment. The reducing cffect of SP on γ-ray-induced micronucleus frequency was blocked by N-acetyl-5-hydroxytryptaminc and methotrexate, which are inhibitors of BH 4 biosynthesis. These results suggest that intracellular BH 4 biosynthesized from sepiapterin is involved in the reducing effect of SP on γ-ray-induced micronucleus frequency. To examine possible mechanisms underlying this action, changes of intracellular BH 4 concentration after sepiapterin treatment were examined. Intracellular BH 4 concentration peaked at 6 h and was maintained at a high level until 24 h after sepiapterin treatment. However, the γ-ray-induced micronucleus frequency at 6 h post-treatment with sepiapterin showed no change in comparison with that after irradiation alone. These results indicate that the reducing effect of BH 4 on γ-ray-induced micronucleus frequency is not necessarily due to antioxidant activity, but may involve other mechanisms.