An antiprotozoal agent pyrimethamine is a potent clastogen that induces structural chromosome aberrations and micronuclei in cultured Chinese hamster (CHL) cells in vitro. Our previous study on the compound, however, demonstrated no significant induction of micronuclei in the mouse bone marrow micronucleus test, even with dosings on 4 consecutive days. In the present study, we investigated the clastogenicity of pyrimethamine in the rat bone marrow micronucleus test. An obvious dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs) was observed when the compound was given 3 times by oral gavage at 20–120 mg/kg/day. The MNPCE frequency at the highest dose was 80 times that of the control group. Single dose of pyrimethamine at 80 and 160 mg/kg also significantly induced MNPCEs. Thus, pyrimethamine showed species-specific effects in rodent bone marrow micronucleus assays. Our finding implies that micronucleus tests using not only mouse, but also rat, may be necessary for the evaluation of clastogens detected in in vitro cytogenetic studies.