Acylphosphatase purified from heart muscle actively hydrolyzes the phosphoenzyme intermediate of cardiac sarcoplasmic reticulum Ca(2+)-ATPase. This effect was evident with acylphosphatase concentrations (up to 100 units/mg sarcoplasmic reticulum protein) that fall within the physiological range, and the low value of the apparent Km, on the order of 10(-7)M, suggests a high affinity towards this special substrate. Moreover, acylphosphatase addition to sarcoplasmic reticulum vesicles significantly enhanced the rate of Ca(2+)-dependent ATP hydrolysis. Maximal stimulation, observed with 100 units/mg vesicular protein, resulted in an ATPase activity which was about two folds over basal value. The same acylphosphatase concentration increased at a similar extent the rate of ATP driven Ca2+ influx into sarcoplasmic reticulum vesicles. Taken together these findings lead to suppose that acylphosphatase, owing to its hydrolytic activity, induces an accelerated turnover of the phosphoenzyme intermediate, whence an overall stimulation of heart sarcoplasmic reticulum Ca2+ pump, affecting both ATP hydrolysis and Ca2+ influx.