The generation of reactive oxygen species (ROS) has been implicated in the regulation of sperm capacitation and acrosome reaction; however, the mechanisms underlying this regulation remain unclear. To examine the cellular processes involved, we studied the effect of different concentrations of hydrogen peroxide (H₂O₂) on protein tyrosine phosphorylation under various conditions. Treatment of spermatozoa with H₂O₂ in medium without heparin caused a time- and dose-dependent increase in protein tyrosine phosphorylation of at least six proteins in which maximal effect was seen after 2 h of incubation with 50 μM H₂O₂. At much higher concentrations of H₂O₂ (0.5 mM), there is significant reduction in the phosphorylation level, and no protein tyrosine phosphorylation is observed at 5 mM H₂O₂ after 4 h of incubation. Exogenous NADPH enhanced protein tyrosine phosphorylation similarly to H₂O₂. These two agents, but not heparin, induced Ca² -dependent tyrosine phosphorylation of an 80-kDa protein. Treatment with H₂O₂ (50 μM) caused approximately a twofold increase in cAMP, which is comparable to the effect of bicarbonate, a known activator of soluble adenylyl cyclase in sperm. This report suggests that relatively low concentrations of H₂O₂ are beneficial for sperm capacitation, but that too high a concentration inhibits this process. We also conclude that H₂O₂ activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation.