Formation of 8-hydroxyguanine within calf thymus DNA has been studied after exposure to uv-H2O2 as a hydroxyl free radical generating system. Using high-pressure liquid chromatography with electrochemical detection, the amount of 8-hydroxy-2-deoxyguanosine (8-OHdG) was measured. The 8-OHdG content of uv-exposed DNA increased linearly with increasing H2O2 levels up to 0.03%, above which the rate of increase was less than linear. All hydroxyl free radical scavengers studied (mannitol, ethanol, thiorea, and salicylate), if present in the system when DNA was exposed to uv-H2O2, caused a decrease in the amount of 8-OHdG formed. Thiourea when incubated with damaged DNA caused a loss of 8-OHdG when it was an integral part of DNA. In contrast, thiorea did not react with the nucleoside free in solution. Reduced glutathione did not cause a decrease of 8-OHdG, either when it was an integral part of DNA, or as the free nucleoside in solution. (Copyright (c) 1988 by Academic Press, Inc.)