Escherichia coli possesses 2 types of O6-methylguanine-DNA methyltransferases, one inducible and the other constitutive. These enzymes are coded by the ada and the ogt genes, respectively. Using a synthetic ogt-specific probe, we mapped ogt at 29.4 min, near the 5'-flanking region of the nirR gene, on the E. coli chromosome. To elucidate the roles of the 2 types of methyltransferases in DNA repair, we constructed mutant strains which lack either one or both of the genes. In either the ada+ or the ada- background, the ogt mutation had no effect on cell survival after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. On the other hand, ada- ogt- cells were more prone to mutation as compared to the ada- ogt+ cells exposed to MNNG. The frequency of spontaneous mutation of cells defective in either one or both of the genes was the same, however, the introduction of the ogt+ plasmid into the cells produced a 2-3-fold decrease in the frequency of spontaneous mutation. O6-Methylguanine-DNA methyltransferases appear to eliminate premutagenic DNA lesions not only from cells exposed to alkylating agents but also from those grown in the absence of the agents.