Acylphosphatase purified from cardiac muscle actively hydrolyzes the phosphoenzyme intermediate of heart sarcolemma Na+,K(+)-ATPase. This effect occurred with acylphosphatase amounts (up to 800 units/mg membrane protein) that fall within the physiological range and the low value of the apparent Km (0.69 x 10(-7) M) indicates a considerable affinity of the enzyme towards this specific substrate. Acylphosphatase addition to purified sarcolemmal vesicles significantly increased the rate of Na+,K(+)-dependent ATP hydrolysis. Maximal stimulation, observed with 800 units/mg protein, resulted in an ATPase activity which was about 2-fold over basal value. The same acylphosphatase amounts significantly stimulated, in a similar and to an even greater extent, the rate of ATP driven Na+ transport into sarcolemmal vesicles. These findings lead to suppose that an accelerated hydrolysis of the phosphoenzyme may result in an enhanced activity of heart sarcolemmal Na+,K+ pump, therefore suggesting a potential role of acylphosphatase in the control of this active transport system.