The mutagenicity of peroxyl radicals, important participants in lipid peroxidation cascades, was investigated using a plasmid-based mutational assay system. Double-stranded pSP189 plasmids were incubated with a range of concentrations of the water-soluble peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH). Following replication in human Ad293 cells, the plasmids were screened for supF mutations in indicator bacteria. Exposure to peroxyl radicals caused strand nicking and a decrease in transfection efficiency, which was accompanied by a significant increase in supF mutants. Each of these effects was abolished in the presence of the water-soluble vitamin E analogue Trolox. Automated sequencing of 76 AAPH-induced mutant plasmids revealed that substitutions at G:C base pairs were the most common changes, accounting for 85.5% of all identified mutations. Of these, most comprised G:C-->T:A transversions (53.5%), with lesser contributions by G:C-->A:T transitions (23.9%) and G:C-->C:G transversions (22.5%). Collectively, these data confirm our previous findings concerning the spectrum of mutations produced upon bacterial replication of peroxyl radical-damaged phage DNA and extend them by showing that such damage has mutagenic consequences during replication in more complex eukaryotic systems.