The enzyme Na+,K+-ATPase creates the transmembrane Na+ gradient that is of vital importance for functioning of all eukaryotic cells. Na+, K+-ATPase can be phosphorylated by protein kinase A (PKA) and protein kinase C (PKC), and these sites of phosphorylation have been identified. In the present study, we have examined the physiological significance of PKC phosphorylation of rat Na+,K+-ATPase. In COS cells transfected with wild type rat Na+,K+-ATPase alpha1, intracellular Na+ was higher and pH was lower than in cells transfected with rat Na+,K+-ATPase alpha1 in which the PKC phosphorylation site, Ser-23, had been mutated into alanine. Phorbol dibutyrate inhibited Na+,K+-ATPase-dependent ATP hydrolysis and Rb+ uptake in cells expressing wild type Na+,K+-ATPase but not in cells expressing S23A Na+,K+-ATPase. Cells expressing the S23A mutant had a more rounded appearance and attached less well to fibronectin than did untransfected cells or cells transfected with wild type rat Na+, K+-ATPase alpha1. These results indicate a functional role for PKC-mediated phosphorylation of rat Na+,K+-ATPase alpha1 and suggest a connection between this enzyme and cell adhesion.