Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na⁺,K⁺-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na⁺,K⁺-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na⁺,K⁺-ATPase α-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na⁺,K⁺-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na⁺,K⁺-ATPase α-subunit. This suggests that phosphorylation does not directly stimulate Na⁺,K⁺-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na⁺,K⁺-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na⁺,K⁺-ATPase activity as cells expressing the wild-type rodent α-subunit, but PMA stimulation of Na⁺,K⁺-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the α-subunit by stimulation of PKC-β, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the α-subunit are essential for PMA stimulation of Na⁺,K⁺-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na⁺,K⁺-ATPase is the result of an increased number of Na⁺,K⁺-ATPase molecules in the plasma membrane.