Oligodeoxynucleotides, modified site-specifically with 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG), 7,8-dihydro-8-oxoadenosine (8-oxodA) and 6-O-methyldeoxyguanosine (O6medG), were used as templates for DNA synthesis in primer-extension reactions catalysed by extracts prepared from human (HeLa) cells, simian kidney (COS-7) cells and various mouse tissues. Fully-extended reaction products were analysed by two-phase polyacrylamide gel electrophoresis (Shibutani, Chem. Res. Toxicol. 6, 625, 1993). Using extracts prepared from HeLa or COS-7 cells, dAMP was preferentially incorporated opposite 8-oxodG; dTMP was incorporated opposite 8-oxodA and dTMP, accompanied by small amounts of dCMP, was incorporated opposite O6medG. Translesional synthesis was strongly inhibited by N-ethylmaleimide and partially inhibited by N-butylphenyl-dGTP. This model system can be used to predict the mutagenic potential of selectively-damaged DNA in mammalian cells.