Mouse spermatozoa released from the cauda epididymidis underwent spontaneous lipid peroxidation during aerobic incubation at 37 degrees C in medium containing 113 mM NaCl, 0.4 mM EDTA, and 15 mM sodium phosphate ( NTPC ). The rate of lipid peroxidation, as measured by malonaldehyde production, was 0.045 nmol malonaldehyde/h per 10(3) cells. The motility of these cells declined with time in medium NTPC ; the percent spermatozoa showing no motility increased linearly with production of malonaldehyde. All flagellar activity stopped at 0.80 nmol malonaldehyde/10(8) cells, independent of the malonaldehyde production rate. Spermatozoa suspended in NTPC at 24 degrees C produced O(2), with an intrinsic rate of 1.96 nmol/min per 10(8) cells; this increased to 3.80 nmol/min per 10(8) cells in 10 mM cyanide. Mouse sperm contain 3.5 U/10(8) cells of superoxide dismutase activity, 91% of which is sensitive, and 9% of which is insensitive, to cyanide inhibition. Mouse sperm also produce H2O2, all of which can be attributed to the action of superoxide dismutase on O(2) produced. Mouse sperm contain high levels of glutathione and of glutathione reductase and peroxidase activities, implicating the glutathione system as the major protective enzyme system against cell damage by autoxidation. This is in contrast to rabbit spermatozoa, which have little endogenous glutathione and rely on superoxide dismutase as protective enzyme against peroxidative damage.