The mutagenic activity of a series of longer chain O6-n-alkylguanine residues (O6-n-propyl, O6-n-butyl, O6-n-octyl) has been analyzed using a plasmid molecule (pUC 9) in which single O6-alkylguanines were positioned in the unique Pstl recognition site by shot gun ligation (Nucleic Acids Res. 13, 3305-3316 (1985)) of overlapping synthetic oligonucleotides. After transfection of these vectors into E. coli cells having normal DNA repair systems, progeny plasmids were produced, of which 2.6%, 2.8% and 4.3% were mutated in their Pstl site when containing O6-n-propylguanine, O6-n-butylguanine, O6-n-octylguanine, respectively. DNA sequence analysis of mutant plasmid genomes revealed that O6-n-propylguanine and O6-n-butylguanine induced exclusively G-->A transitions located specifically at the preselected site. O6-n-octylguanine induced apart from G-->A transitions (70%) also targeted G-->T transversions (30%). These results indicate that the mutation frequency of longer chain O6-alkylguanines can be substantial in cells with normal repair systems and that the mutation pattern depends on the nature of the alkyl group.