It has been reported that exogenous alkylated purines, such as O6-methylguanine (O6meG), induce aneuploidy in mammalian cells. It is shown here that the aneugenic effect of O6meG, evidenced by its ability to induce micronuclei in rodent cells, is dependent on its conversion to O6-methyl-guanosine-5'-monophosphate (O6me-5'-GMP) by hypoxanthine-guanine phosphoribosyl transferase (HPRT). This conclusion, in contrast with previous in vitro data showing that O6meG does not seem to be a substrate for HPRT, was based on the following observations: 1) O6meG did not induce micronuclei in HPRT-deficient Chinese hamster cells, but did induce micronuclei in HPRT-proficient cells, and in mouse cells partially or totally deficient in adenine phosphoribosyl transferase; 2) O6meG was not metabolized in HPRT-deficient cells, while in wild-type cells a number of metabolites were detected by high performance liquid chromatography (HPLC) analysis of cold acid extracts, one of them coeluting with O6me-5'-GMP used as a marker; 3) when de novo synthesis of purine nucleotides was inhibited by aminopterin, O6meG sustained the growth of HPRT-proficient, but not of HPRT-deficient, cells; and 4) when HPRT-deficient cells were treated with liposomes charged with O6me-5'-GMP, induction of micronuclei was shown. The finding that methylated guanine exerts its aneugenic action through methylated nucleotide(s) provides an important, though indirect, support to the hypothesis that alkylating agents may induce aneuploidy via nucleotide pool alkylation.