Site-specific mutagenesis by O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobGua), a product of DNA pyridyloxobutylation by metabolites of the tobacco-specific nitrosamines N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), was studied in Escherichia coli strain DH10B and human kidney cells (293) when the modified base was incorporated in either a double-stranded or a gapped shuttle vector. In the repair-competent E. coli strain, less than 3% of the colonies produced by double-stranded vectors harboring the modified base were mutant whereas 96% were mutant when DH10B cells were transformed with modified gapped vectors. By contrast, transformation of DH10B cells with plasmids derived from O6-pobGua-containing double-stranded and gapped vectors previously replicated in 293 cells produced 7 and 16% mutant colonies, respectively. These percentages increased to 42 and 82%, respectively, when the 293 cells were pretreated with O6-benzylguanine to inactivate the O6-alkylguanine-DNA alkyltransferase protein. These findings confirm that the adduct is readily repaired by the human O6-alkylguanine-DNA alkyltransferase in both double-stranded and gapped vectors and suggest that it is also highly mutagenic in both human cells and E. coli. In the E. coli strain, the adduct produced exclusively G → A transition mutations although in human 293 cells it also produced G → T transversions and more complex mutations in addition to G → A transitions. These data suggest that O6-[4-oxo-4-(3-pyridyl)butyl]guanine can contribute significantly to the mutagenic risk posed by exposure to both NNN and NNK in tobacco smoke.