The reaction between xanthine and xanthine oxidase results in the univalent and divalent reduction of dioxygen to generate superoxide (O2-.) and hydrogen peroxide (H2O2), respectively. With the aid of this system, the direct effect of reactive oxygen species (ROS) on human sperm function has been investigated. A protocol involving the addition of xanthine oxidase to the reaction mixture at 0 and 15 min resulted in a loss of motility involving every component of sperm movement examined. Lower doses of xanthine oxidase, which did not influence sperm motility, were also found to suppress the competence of human spermatozoa to exhibit oocyte fusion in response to the ionophore, A23187. The reactive oxygen species responsible for the disruption of human sperm function was not influenced by the presence of superoxide dismutase (SOD) or scavengers of hypochlorous acid or hydroxyl radicals. However, the cytotoxic species was shown to be extremely stable and could be completely eliminated by catalase, which selectively eliminates H2O2. Confirmation that it is H2O2, and not O2-., which is cytotoxic to human spermatozoa was obtained in studies in which the direct addition of this oxidant was shown to influence both the movement of human spermatozoa and their competence for oocyte fusion. These results carry implications for the diagnosis of defective sperm function and the design of optimized culture media for the treatment of male factor infertility.