To clarify the involvement of O6-methylguanine (O6-MeG) in mutagenesis, we have been trying to isolate Mer+ cells from a HeLa S3 Mer- cell line, and to compare the mutation frequencies between the cell lines. We previously isolated the N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-resistant cells, MR10-1, from HeLa S3 Mer- cells. However, the MR10-1 cells still had only a little O6-MeG-DNA methyltransferase (MT) activity. In the present study, we have isolated two 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU)-resistant cells, ACr41 and ACr42, from the MR10-1 cells. The two ACr cells had increased MT activities. The ACr cells were also significantly more resistant to 1-(2-chloroethyl)-1-nitrosourea and slightly more resistant to MNNG than the MR10-1 cells. When the mutation frequencies were tested at the hypoxanthine-guanine phosphoribosyl transferase and ouabain loci in these cell lines, the two ACr cells were more resistant to the mutagenic effect of MNNG than the MR10-1 cells. These results show the linkage between the resistance to the cytotoxic effect of nitrosourea compounds and MT activity, and strongly support the hypothesis that O6-MeG is the main pre-mutagenic lesion induced by MNNG.