Rabbit spermatozoa released from the cauda epididymidis into Tris phosphate medium containing KCl or NaCl and 0.4 mM EDTA underwent spontaneous lipid peroxidation during aerobic incubation at 37 degrees C. In the medium containing 130 mM K+ and O mM Na+ (KTP), the rate of lipid peroxidation, as measured by malonaldehyde production, proceeded at a linear rate of 0.045 nmol malonaldehyde/h per 10(8) cells for 22 h. The motility of these spermatozoa declined with time in medium KTP, with 40% initial forward motility decreasing to zero in 4 h and initial 60% flagellar beating ceasing after 12 h. The percent inert spermatozoa showing no flagellar motion in KTP increased linearly with production of malonaldehyde; all flagellar activity stopped at 0.5 nmol malonaldehyde/10(8) cells. In the Tris phosphate medium containing 120 mM Na+ and 10 mM K+ (NTP), the percentage of sperm showing forward motility was close to 100% and this declined to 60% after 16 h aerobic incubation. Flagellar beating was not observed. In medium NTP, the rate of lipid peroxidation was 0.0056 nmol malonaldehyde/h per 10(8) cells, eightfold lower than that observed in KTP. The same linear correlation between malonaldehyde production and percent inert sperm was found as for KTP: 0.5 nmol malonaldehyde/10(8) cells also corresponded to cessation of flagellar motion. The dependence of motility maintenance on K+ concentration in Tris phosphate medium containing (Na+ + K+)=130 mM showed maximal maintenance at 10 mM K+, with a decline at 0 mM K+ and steep decline at K+ concentrations greater than 30 mM. This strong dependence of rabbit sperm peroxidation on ionic composition of the medium is suggested to involve perturbation of the equilibrium between O2 .- and its conjugate acid species being the agent of peroxidation.