In vitro incubation of rat microsomal lipids with NADPH and Fe3+ in the presence of cytochrome P450 reductase produces lesions in double-stranded pZ189 plasmid DNA, the mutagenic potential of which was analyzed after transfection into Escherichia coli host cells that had been induced for SOS functions by ultraviolet irradiation. The extent of lipid peroxidation, when monitored by the formation of thiobarbituric acid reaction substances, was increased with increased addition of lipids in the reaction mixture. Mutagenesis was determined with the forward mutation assay using the supF gene of pZ189 as a target. When treated pZ189 DNA was used to transfect host cells, a seven-fold increase in mutation frequency for SOS uninduced hosts and a 12-fold increase in mutation frequency for SOS induced hosts was observed at 50% survival compared to that observed with untreated DNA. Sequence analysis shows that incubation of pZ189 DNA in the lipid peroxidation reaction mixture results mostly in single base substitutions, the most frequent base change being G:C-->C:G transversion, followed by G:C-->T:A transversion. The fact that, in the SOS induced hosts, the spectrum obtained by lipid peroxidation is similar to that of hydrogen peroxide suggests the possible involvement for mutagenesis of superoxide produced during lipid peroxidation, but not lipid peroxidation end products such as aldehyde or alkane. Treatment of pZ189 DNA with increasing extents of lipid peroxidation did not yield increasing formation of 8-hydroxyguanine. The results suggest that the origins of G:C-->C:G and G:C-->T:A transversions may be (an) as yet unidentified lesion(s) generated by lipid peroxidation.