To compare mutagenesis by O6-methylguanine (m6G), O4-methylthymine (m4T) and O6-ethylguanine (e6G), and assess their genotoxicity in Escherichia coli, double-stranded and gapped plasmids were constructed containing a single m6G, e6G or m4T in the initiation codon (ATG) of a lacZ' gene. Modified base induced mutations were scored by the loss of lacZ' activity on X-gal-containing media resulting in formation of white or sectored (mutant) rather than blue (non-mutant) colonies. Genotoxicity experiments with gapped plasmids containing the modified bases indicated that m4T produced a greater number of bacterial colonies than m6G or e6G. m4T was more mutagenic (45% mutant colonies) than m6G (6%) or e6G (11%) in repair competent (w.t.) E. coli when incorporated in double-stranded plasmids. In gapped plasmids, m4T produced 99% mutant colonies (as was observed previously for e6G) in both w.t. E. coli or E. coli deficient in both O6-alkylguanine-DNA alkyltransferases as well as methylation-directed mismatch repair (ada(-)-ogt(-)-mutS[-]). m6G in gapped plasmids produced 62% mutant colonies in w.t. E. coli, but this percentage increased to 94% in the ada(-)-ogt(-)-mutS(-) strain. In double-stranded plasmids both m4T and m6G produced very similar distributions of mutant and non-mutant colonies in the ada(-)-ogt(-)-mutS(-) strain. These observations led to the conclusion that differences in the mutagenicity of m6G and m4T in w.t. E. coli were a result of preferential repair of m6G compared to m4T by alkyltransferase and mismatch repair mechanisms, and did not reflect differences in their respective coding efficiency or their inherent obstructiveness to DNA synthesis as was observed with e6G. The combination of alkyltransferase and mismatch repair was concluded to be primarily responsible for the apparent genotoxicity of m6G compared to m4T in double-stranded plasmids.