Methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR), slightly induced micronuclei in bone marrow and peripheral blood cells, and this induction was enhanced by multiple treatments with the drug. Furthermore, we have suggested that the multiple-dose effect on the induction of micronuclei by MTX might be explained by intracellular accumulation of the drug, resulting in an enhancement of DHFR inhibition. An imbalance or decrease in the deoxyribonucleotide (dNTP) pool would be generated by this enzyme inhibition. Therefore, we attempted to determine the level of the dNTP pool in mouse bone marrow cells. The levels of three dNTPs (dTTP, dATP, dGTP) as determined by HPLC were only 1/10-1/40 of the levels previously found in mammalian cell lines, but dCTP levels could not be determined precisely because they approached the limits of detectability. The levels of dTTP, dATP and dGTP in mouse bone marrow cells 3 h after four injections of MTX (4 mg/kg/day) decreased to 21.2%, 47.0% and 38.1%, respectively, of those in the control group. The level of dTTP 3 h after four injections of 100 mg/kg of the drug decreased to almost 0%. The results of alkaline elution assays suggested that alkaline-labile sites were generated in mouse bone marrow cells 6 h after four injections of MTX (4 mg/kg). These findings suggest that the multiple-dose effects of MTX on micronucleus induction in mouse bone marrow cells may be explained by the decrease in the dNTP pool and subsequent generation of alkaline-labile sites (possibly apurinic/apyrimidinic sites).