The mutagenic activity of a series of longer chain O⁶-n-alkylguanine residues (O⁶-n-propyl, O⁸-n-butyl, O⁶-n-octyl) has been analyzed using a plasmid molecule (pUC 9) in which single O⁶alkylguanines 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-npropylguanine, O⁶-n-butylguanine, O⁶-n-octylguanine, respectively. DNA sequence analysis of mutant plasmid genomes revealed that O⁸-n-propylguanine and O⁶-nbutylguanine induced exclusively G–A transitions located specifically at the preselected site. O⁶-noctylguanine 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.