We have investigated whether the presence of a DNA repair enzyme, O6-methylguanine-DNA-methyltransferase (MGMT), affects the nature of spontaneous mutations in a mammalian cell line. We compared spontaneous mutations in the adenine phosphoribosyl transferase gene of a Chinese hamster ovary (CHO) cell line that expressed 14,000 MGMT molecules/cell with those in the parental CHO cells lacking this DNA repair activity. The mutation rate/cell/generation of the two CHO cell lines did not differ significantly. However, DNA sequence analysis of spontaneous mutations in the MGMT-proficient CHO cell line revealed a complex picture. No significant difference from the parental CHO cells was found in the number or type of deletions, frameshifts, multiple substitutions, or insertions. The frequency of G:C to T:A transversions was elevated in MGMT-proficient CHO cells. Expression of the enzyme considerably reduced G:C to A:T transitions (25% versus 8.3%). This latter result is the first evidence that this protein is active on an endogenous source of O6-methylguanine that is normally responsible for spontaneous G:C to A:T transition mutations.