The mechanism of activation of renal Na+-K+-ATPase was studied in rats 2 wk after unilateral nephrectomy. The increase in enzyme specific activity was confined to the outer medulla and occurred without changes in the cellular contents of RNA or protein. Enzyme activation was accompanied by increases in the levels of the phosphorylated intermediate with little or no change in the apparent turnover numbers of the reaction. The specific activity of the ouabain-sensitive p-nitrophenylphosphatase also increased by uninephrectomy but to a larger extent than did Na+-K+-ATPase. Kinetic studies demonstrated an increase in Vmax for ATP, sodium, and potassium, and small increases in Km for ATP and K1/2 for potassium. There was no change in the activation energies or phase transition temperature to indicate alterations in the membrane environment of Na+-K+-ATPase. Adrenalectomy did not adversely affect activation. These results indicate that activation of renal Na+-K+-ATPase after reduction of renal mass occurs mainly by an increase in the number of sodium pump sites.