Receptor-mediated activation of the sodium pump has been noted in several intact tissues. To test the hypothesis that this may be due to the direct effects of the second messenger diacylglycerols on the pump, we studied the effects of various long-chain acylglycerols on the purified Na+/K(+)-ATPase. With optimal ATP, acylglycerols had no effect on enzyme activity. When ATP was suboptimal, tri- and diacylglycerols had no effects, but monoacylglycerols caused up to 3-fold increase in ATPase activity. Using sealed vesicles of red cell membranes and cardiac sarcolemma, stimulation of the ion transport function of the enzyme by monoacylglycerols in the presence of suboptimal ATP was also shown. Since the sodium pump may not be saturated with ATP in the intact cell, the possibility arises that monoacylglycerols are the second messengers for the receptor-mediated regulation of the pump.