Human spermatozoa were exposed to the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to stimulate endogenous production of reactive oxygen species (ROS). They were incubated under a gas phase of 5% CO2/90% N2/5% O2, or 5% CO2/95% air (20% O2) to investigate whether a lower than atmospheric oxygen tension in the gas phase of the incubator limited the endogenous production of ROS by human spermatozoa and thus was able to reduce the cytotoxic effects of ROS on sperm function. Exposure of human spermatozoa or exogenous NADPH induced an 8-fold higher production of superoxide anion under ambient vs. low oxygen tension. This marked difference in the stimulation of superoxide anion generation was associated with significantly different sperm motility parameters, according to the oxygen tension in the gas phase of the incubator. Whereas under 5% oxygen the percentage of motile spermatozoa was unaffected by the presence of NADPH, all of the motility parameters recorded under an atmosphere of 5% CO2 in air were dramatically affected, not only compared to their respective controls, but also compared to the motility parameters observed under low oxygen tension. The presence of superoxide dismutase plus catalase protected spermatozoa against the toxic effects of NADPH, confirming a cause/effect relationship between the increased superoxide production and reduced sperm function. Even at a concentration of NADPH which did not alter the percentage of motile spermatozoa, hyperactivated motility and acrosome reaction were significantly lower under an atmosphere of 5% CO2 in air compared to a gas phase of 5% CO2/90% N2/5% O2. These results suggest that there is an advantage in using 5% O2 rather than 20% O2 in the gas phase of the incubator to prevent the excessive production of ROS by spermatozoa and related alterations of sperm functions. This may be of clinical value in fertilization programmes.