This work examines the mutagenic activity of O6-methylguanine (O6MeGua), a DNA adduct formed by certain carcinogenic alkylating agents. A tetranucleotide, 5'-HOTpm6GpCpA-3', was synthesized and ligated into a four-base gap in the unique Pst I site of the duplex genome of the E. coli virus, M13mp8. The double-stranded ligation product was converted to single-stranded form and used to transform E. coli to produce progeny phage. The mutation frequency of O6MeGua was defined as the percentage of progeny phage with mutations in their Pst I site, and this value was determined to be 0.4%. To determine the impact of DNA repair on mutagenesis, cellular levels of O6MeGua-DNA methyltransferase (an O6MeGua-repair protein) were depleted by treatment of host cells for virus replication with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) prior to viral DNA uptake. In these host cells, the mutation frequency due to O6MeGua increased markedly with increasing MNNG dose (the highest mutation frequency observed was 20%). DNA sequence analysis of mutant genomes revealed that in both MNNG treated and untreated cells, O6MeGua induced exclusively G to A transitions.