Previous work suggested that arsine- (AsH3-) induced hemoglobin (Hb02) damage may lead to hemolysis (Hatlelid et al., 1996). The purpose of the work presented here was to determine whether reactive oxygen species are formed by AsH3in solution, in hemoglobin solutions, or in intact red blood cells, and, if so, to determine whether these species are responsible for the observed hemoglobin damage. Hydrogen peroxide (H202) was detected in aqueous solutions containing AsH3and Hb02or AsH3alone but not in intact red blood cells or lysates. Additionally, high-activity catalase (19,200 U/ml) or glutathione peroxidase (68 U/ml) added to solutions of Hb02and AsH3had only a minor protective effect against AsHyinduced damage. Further, the differences between the visible spectra of AsH3-treated Hb02and H202-treated HbO2indicate that two different degradative processes occur. The presence of superoxide anion (Of) was measured by O2-dependent reduction of nitro blue tetrazolium (NBT). The results were negative for Of. Exogenous superoxide dismutase (100 ug/ml) did not affect AsH3-induced Hb02spectral changes, nor did the hydroxyl radical scavengers, mannitol, and DMSO (20 mM each). The general antioxidants ascorbate (<10 mM) and glutathione (<1 mM) also had no effect. These results indicate that the superoxide anion and the hydroxyl radical COH) are not involved in the mechanism of AsH3-induced Hb02damage. The results also indicate that although AsH3contributes to H202production in vitro, cellular defenses are adequate to detoxify the amount formed. An alternative mechanism by which an arsenic species is the hemolytic agent is proposed.