In alveolar epithelial cells, G‐protein coupled‐receptors agonists (GPCR) induce the recruitment of the Na,K‐ATPase to the plasma membrane. Here we report that for the recruitment of the Na,K‐ ATPase to occur, dephosphorylation of its α1‐subunit at serine 18 is necessary, as demonstrated by in vitro phosphorylation, mutation of the serine 18 to alanine, and use of a specific phospho‐antibody. Several approaches strongly suggest dephosphorylation to be mediated by protein phosphatase 2A (PP2A): 1) Na,K‐ ATPase dephosphorylation and recruitment were prevented by okadaic acid (OA); 2) the Na,K‐ATPase α1‐subunit is an in vitro substrate for PP2A; and 3) glutathione S‐transferase (GST)‐fusion proteins binding assays demonstrate a direct interaction between the catalytic subunit of PP2A and the first 90 amino acids of the Na,K‐ATPase α1‐subunit. Finally, GPCR agonists induced a rapid translocation of PP2A from the cytosol to the membrane fraction, which corresponded with increased coimmunoprecipitation and colocalization of PP2A and the Na,K‐ATPase. Accordingly, we provide evidence that GPCR agonists promote PP2A translocation to the membrane fraction, leading to the dephosphorylation of the Na,K‐ATPase α1‐subunit at the serine 18 residue and its recruitment to the cell plasma membrane, which is of biological and physiological importance.—Lecuona, E., Dada, L. A., Sun, H., Butti, M. L., Zhou, G., Chew, T.‐L., Sznajder, J. I. Na,K‐ ATPase α1‐subunit dephosphorylation by protein phosphatase 2A is necessary for its recruitment to the plasma membrane. FASEB J. 20, E2146–E2155 (2006)