O6-Methylguanine-DNA methyltransferase (MT)-proficient Chinese hamster ovary cells were grown in the presence of low, gradually increasing levels of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with the aim of selecting MNNG-resistant cell lines. Six resistant clones with two levels of resistance were isolated. A 3-fold increase in survival was observed in clones 13, 14 and 15 and a greater than 10-fold increase in clones A, B and C. Cross resistance to N-methyl-N-nitrosourea but not to mitomycin C was observed. By comparison with the parental MT-proficient cells, MT activity was doubled in two resistant clones (13 and B) irrespective of their resistance levels. DNA glycosylase activity responsible for the removal of 7-methylguanine and 3-methyladenine showed similar levels in resistant clones 13 and B, in the MT-proficient cells and in the original MT-deficient cells. Alkylation-induced DNA damage, as measured by alkaline elution at the same MNNG dose, was higher in clones 13 and B than in the parental cells. The induction of sister chromatid exchanges by MNNG was inversely related to the resistance levels, thus paralleling the induction of cytotoxicity. These results suggest the existence in Chinese hamster ovary cells of at least two independent functions which control resistance to methylating agents, one possibly being the capacity to repair O6-methylguanine.