Oxidative stress is an important signal for apoptosis to start. So far the mitochondrial respiratory chain has been considered as the major, if not the only, cause of such stress. Here we report that this is not the case. Xanthine oxidase, a O2(-) and H2O2 generating enzyme which is important in causing significant oxidative stress in the cytosol, is also present in the mitochondrial fraction of rat mammary gland. After weaning, during the involution of the mammary gland, massive apoptosis occurs. Mitochondrial xanthine oxidase activity increases and high mitochondrial H2O2 production takes place. Inhibition of xanthine oxidase activity by allopurinol, a specific inhibitor of xanthine oxidase activity, slows down the involution of the mammary gland due to the decrease in the number of apoptotic cells and prevents the production of H2O2 that occurs during apoptosis. Thus, mitochondrial xanthine oxidase by means of its production of O2(-) and H2O2 can maintain the apoptotic machinery during the involution of the mammary gland after weaning and could be considered necessary to maintain the apoptotic cascade during the physiological involution of tissues. Oxidative stress generated during apoptosis by mitochondria is not only due to the respiratory chain.