Rabbit spermatozoa from the cauda epididymis produced 0.7-0.8nmol of H(2)O(2)/min per 10(8) cells at cell concentrations below 10(7) cells/ml with linear dependence on cell concentration. Above 2 x 10(7) cells/ml, the rate again became linear with cell concentration but decreased to 0.1-0.2nmol/min per 10(8) cells. Spermatozoa treated with amphotericin B, which makes the plasma membrane highly permeable to low-molecular-weight compounds, showed a similar dependence of H(2)O(2) production rate on cell concentration; below 10(7) cells/ml the rate was 0.3-0.4nmol/min per 10(8) cells; above 2 x 10(7) cells/ml, the rate was 0.1-0.2nmol/min per 10(8) cells. Hypo-osmotically treated rabbit epididymal spermatozoa, a preparation useful for studying mitochondrial function in sperm [Keyhani & Storey (1973) Biochim. Biophys. Acta305, 557-565] produced 0.1-0.2nmol/min per 10(8) cells in the absence of added substrates. The dependence of rate on cell concentration was linear from 10(7) to 2.2 x 10(8) cells/ml. This endogenous rate was unaffected by rotenone, but stimulated 4-fold by antimycin A. Addition of the mitochondrial substrates lactate plus malate increased the rate of H(2)O(2) production to 0.3nmol/min per 10(8) cells. The decreased rate of H(2)O(2) production observed with intact sperm at high cell concentrations is attributed to reaction of H(2)O(2) with the cells, possibly with the plasma membrane, which is lost after hypo-osmotic treatment. Rabbit spermatozoa have glutathione peroxidase and glutathione reductase activities, but these seem to play little role in removal of H(2)O(2) generated. The rate at low cell concentration is taken to be the unperturbed rate. The sources of H(2)O(2) production in rabbit spermatozoa have been tentatively resolved into a low-molecular-weight component, lost after amphotericin treatment, a mitochondrial component and a rotenone-insensitive component that has not been identified.