Functional properties of Na,K-ATPase can be modified by association with FXYD proteins, expressed in a tissue-specific manner. Here we show that expression of FXYDs in cell lines does not necessarily parallel the expression pattern of FXYDs in the tissue(s) the cells originate from. While being expressed only in lacis cells in the juxtaglomerular apparatus and in blood vessels in kidney, FXYD1 was abundant in renal cell lines of proximal tubule origin (NRK-52E, LLC-PK1, and OK cells). Authenticity of FXYD1 as a part of Na,K-ATPase in NRK-52E cells was demonstrated by co-purification, co-immunoprecipitation and co-localization. Induction of FXYD2 by hypertonicity (500 mOsm with NaCl for 48 h or adaptation to 700 mOsm) correlated with down-regulation of FXYD1 at mRNA and protein levels. The response to hypertonicity was influenced by serum factors and entailed first, dephosphorylation of FXYD1 at Ser68 (1-5 hours), and second, induction of FXYD2a and decrease in FXYD1 with longer exposure. FXYD1 was completely replaced with FXYD2a in cells adapted to 700 mOsm and showed a significantly decreased sodium affinity. Thus dephosphorylation of FXYD1 followed by exchange of regulatory subunits is utilized to make a smooth transition of properties of Na,K-ATPase. We also observed expression of mRNA for multiple FXYDs in various cell lines. The expression was dynamic and responsive to physiologic stimuli. Moreover, we demonstrated expression of FXYD5 protein in HEK293 and HeLa cells. The data imply that FXYDs are obligatory rather than auxiliary components of Na,K-ATPase, and their interchangeability underlies responses of Na,K-ATPase to cellular stress.