We have demonstrated that free radicals generated by hydrogen peroxide (H2O2), in the presence of divalent iron (Fe2+) and a chelator (EDTA), oxidize 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG). The 8-OHdG formed by this reaction was isolated and quantitated using reverse-phase HPLC with UV and electrochemical detection. A 1-h incubation of dG with H2O2 caused a 50% increase in 8-OHdG over background, which increased to 100% after 2 h. However, when an H2O2-generating system [glutathione (GSH), Fe2+, EDTA] was used, there was no increase in 8-OHdG yield after the 1-h incubation, but up to a 50% increase over background was observed with GSH after 2-h incubation. Attempts to detect increased levels of 8-OHdG after H2O2- or GSH-treatment of purified calf thymus or rat DNA, or purified Salmonella typhimurium DNA were not successful. This may have been because the treatment procedures used generated 8-OHdG in the control samples at sufficiently high levels to mask any H2O2-induced responses that may have been present. This artifactual production of 8-OHdG has presented a problem in all in vitro studies to date. In contrast, treatment of Salmonella cells (strain TA104) with increasing concentrations of H2O2, caused a doubling in the 8-OHdG yield. GSH-treatment of strain TA104 cells under the same conditions did not result in an increase of 8-OHdG. The study presented here shows that the ubiquitous molecule H2O2 can play a major role in DNA oxidation, mutation, and damage.