The mechanisms by which Pb(2+) induces hemolysis are not completely understood. For this reason, the involvement of oxidative stress in the mechanism of Pb(2+)-induced pre-hemolytic lesion was investigated by exposing RBC to Pb(2+) in vitro and then separating the intact non-hemolysed RBC. Oxidative stress was investigated on human RBCs by tert-butyl hydroperoxide-initiated chemiluminescence method (CL). Our results revealed that lead-induced time and concentration-dependent hemolysis and CL time curves showed a very narrow correlation each other. GSH oxidation to GSSG and the stress index also increased significantly. Treatment of lead-exposed RBC with desferrioxamine, an iron-chelating agent or the chain-breaking antioxidant, Trolox, quenched light emission and inhibited hemolysis dramatically. Mannitol and sodium formate, (*)OH scavengers, on the contrary, did not inhibit CL or hemolysis, significantly. These data indicate that lead-induced lipid peroxide formation is mediated by a metal-driven Fenton reaction but do not support the direct involvement of hydroxyl radicals in this process. By contrast, our results revealed a decrease in light emission and decreased hemolysis in the presence of histidine, a singlet oxygen scavenger. Our results suggest that membrane damage and hemolysis of RBC are mediated by Pb(2+) through free radical reactions and that singlet oxygen plays a significant role in this process.