Plasmids containing a site-specifically incorporated O6-methyl- (m6G), O6-ethyl- (e6G), or O6-benzylguanine (b6G) within the ATG initiation codon of the lacZ' gene were used to transform Escherichia coli that were repair proficient or deficient in one or both of the E. coli O6-alkylguanine-DNA alkyltransferases, the uvr(ABC) excision repair system, the recA-mediated recombination system, or the methylation-directed mismatch repair system. Colonies were scored phenotypically for adduct-induced mutations. With plasmids containing either e6G or b6G, the frequency of adduct-induced mutation was low and independent of the repair proficiency of the strain transformed. Plasmids containing an m6G residue elicited similar responses in all but the mismatch repair-deficient strain. The generally low mutagenicity of all the O6-substituted guanines was interpreted as reflecting an adduct-induced arrest of replication of the modified strand while the unmodified complementary strand was replicated normally. Studies of the involvement of mismatch repair in m6G mutagenesis showed that m6G:T base pairs were more readily processed than m6G:C base pairs, indicating that mismatch repair involving m6G residues occurs after replication. These data support a model in which the E. coli methylation-directed mismatch repair system diverts plasmids containing promutagenic m6G:T base pairs into replication-arrested complexes providing another line of defense against O6-methylguanine mutagenicity in addition to O6-alkylguanine-DNA alkyltransferase repair and excision repair mechanisms.