Mouse and human spermatozoa, but not rabbit spermatozoa, have long been known to be sensitive to loss of motility induced by exogenous H2O2. Recent work has shown that loss of sperm motility in these species correlates with the extent of spontaneous lipid peroxidation. In this study, the effect of H2O2 on this reaction in sperm of the three species was investigated. The rate of spontaneous lipid peroxidation in mouse and human sperm is markedly enhanced in the presence of 1-5 mM H2O2, while the rate in rabbit sperm is unaffected by H2O2. The enhancement of lipid peroxidation, the rate of reaction of H2O2 with the cells, the activity of sperm glutathione peroxidase, and the endogenous glutathione content are highest in mouse sperm, intermediate in human sperm, and very low in rabbit sperm. Inactivation of glutathione peroxidase occurs in the presence of H2O2 due to complete conversion of endogenous glutathione to GSSG: No GSH is available as electron donor substrate to the peroxidase. Inactivation of glutathione peroxidase by the inhibitor mercaptosuccinate has the same effect on rate of lipid peroxidation and loss of motility in mouse and human sperm as does H2O2. This implies that H2O2 by itself at 1-5 mM is not intrinsically toxic to the cells. With merceptosuccinate, the endogenous glutathione is present as GSH in mouse and human sperm, indicating that the redox state of intracellular glutathione by itself plays little role in protecting the cell against spontaneous lipid peroxidation. Mouse and human sperm also have high rates of superoxide production. We conclude that the key intermediate in spontaneous lipid peroxidation is lipid hydroperoxide generated by a chain reaction initiated by and utilizing superoxide. Removal of this hydroperoxide by glutathione peroxidase protects these sperm against peroxidation; inactivation of the peroxidase allows lipid hydroperoxide to increase and so increases the peroxidation rate. Rabbit sperm have low rates of superoxide reaction due to high activity of their superoxide dismutase; lack of endogenous glutathione and low peroxidase activity does not affect their rate of lipid peroxidation. As a result, these sperm are not affected by either H2O2 or mercaptosuccinate. These results lead us to postulate a mechanism for spontaneous lipid peroxidation in mammalian sperm which involves reaction of lipid hydroperoxide and O2 as the rate-determining step.