The possible diurnal variation in cardiac [Ca(2+) + Mg2+]-dependent ATPase (Ca2+ pump) activity and the influence of pinealectomy and melatonin on this enzyme in rat heart have been studied. Lowest levels of cardiac sarcolemmal membrane [Ca(2+) + Mg2+]-dependent ATPase activity were measured in late afternoon in rats kept under a 14:10 light:dark cycle. Late in the dark phase the enzyme activity began to increase with the rise continuing until 0900, 3 hr after light onset. These time-dependent changes in [Ca(2+) + Mg2+]-dependent ATPase activity did not occur in either pinealectomized or light-exposed rats suggesting that melatonin, secreted from the pineal gland during the night, induces the change in [Ca(2+) + Mg2+]-dependent ATPase activity, In vitro studies in which cardiac tissue was incubated in the presence of melatonin over a wide range of doses showed that this indole stimulated the Ca2+ pump. The half-maximal effect of melatonin was observed at a melatonin concentration of 28 ng/ml. These findings suggest that the daily change in [Ca(2+) + Mg2+]-dependent ATPase activity in the sarcolemma of heart tissue is a result of the circadian rhythm in pineal melatonin production and secretion. These findings may be applicable to normal cardiac physiology.