We showed previously that resistance of a series of human fibroblast cell lines to the cytotoxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is highly correlated with their level of O6-methylguanine-DNA-methyltransferase (MT) activity. In the present study, MT activity in normal fibroblasts was decreased to between 40 and 20% of the constitutive level by 15 or 24 h exposure of the cells to exogenous O6-methylguanine (O6-MeG). MT-depleted and non-depleted populations were then challenged with various doses of MNNG and assayed for cytotoxicity and mutagenicity. At every dose the frequency of 6-thioguanine resistant cells induced by MNNG was higher in the MT-depleted populations than in the controls. Since the MT activity in these cells does not remove methyl from the O4 position of thymine, these results strongly support the hypothesis that O6-methylguanine is the principal mutagenic lesion induced by MNNG. Cells with decreased levels of MT were not significantly more sensitive to the cytotoxic effect of MNNG. If O6-methylguanine is a potentially cytotoxic lesion, this lack of increased sensitivity may reflect the fact that regeneration of MT protein occurred rapidly enough to remove these lesions before they resulted in cell death (i.e., inability to form a clone). Consistent with this explanation is the fact that 7 h after removal of the exogenous O6-MeG, the level of MT activity had regenerated to 51% of normal; by 18 h, it was 65% of normal.