Cryopreservation introduces extreme temperature and osmolality changes that impart lethal and sublethal effects on spermatozoa survival. Additionally, evidence indicates that the osmotic stress induced by cryopreservation causes oxidative stress to spermatozoa as well. Our objective was to determine the effect of reactive oxygen species (ROS) on rhesus macaque (Macaca mulatta) sperm function and to determine whether osmotic stress elicits the production of ROS. In the first experiment, the xanthine-xanthine oxidase (X-XO) system was used to generate the ROS superoxide anion (O₂^−·) and hydrogen peroxide (H₂O₂) in the presence or absence of the ROS scavengers superoxide dismutase and catalase, respectively. In the second experiment, osmotic stress was introduced by incubation of spermatozoa in a series of anisosmotic media ranging from 100 to 1000 mOsmol/kg in the presence or absence of the antioxidant alpha-tocopherol. Treatment with the X-XO system resulted in a significant increase in the generation of O₂^−· and H₂O₂ that was detectable using flow cytometry. The ROS generated by the X-XO system was dose dependent, and as the concentration of ROS increased, motility decreased and lipid peroxidation increased while no affect was observed on viability. Incubation of spermatozoa in anisosmotic media also resulted in an increase in O₂^−· generation and lipid peroxidation that was significantly decreased in the presence of the powerful antioxidant alpha-tocopherol. These results clearly indicate that osmotic stress causes oxidative stress in rhesus macaque spermatozoa, which strongly supports the hypothesis that cryopreservation-induced osmotic stress may lead to oxidative cell damage.