Acylphosphatase, purified from human erythrocytes, actively hydrolyzes the phosphoenzyme intermediate of human red blood cell membrane Na+, K(+)-ATPase. This effect occurred with acylphosphatase amounts (up to 10 units/mg membrane protein) that fall within the physiological range. Acylphosphatase addition to erythrocyte membranes resulted in a significant increase in the rate of Na+, K(+)-dependent ATP hydrolysis. Maximal stimulation, observed with 10 units/mg membrane protein, was of about 80% over basal value. The same acylphosphatase amount enhanced of about 40% the rate of ATP driven Na+ transport into inside out red cell membrane vesicles. Taken together these findings suggest a potential role of acylphosphatase in the control of the activity of erythrocyte membrane Na,K pump.