The FXYD protein family consists of several small, single-span membrane proteins that exhibit a high degree of homology. The best known members of the family include the subunit of the Na,K-ATPase and phospholemman (PLM), a phosphoprotein of cardiac sarcolemma. Oher members of the family include corticosteroid hormone-induced factor (CHIF), mammary tumor protein of 8-kDa (Mat-8), and related to ion channels (RIC). The exact physiological roles of the FXYD proteins remain unknown. To better characterize the function of the members of the FXYD protein family, we expressed several members of the family in Madin-Darby canine kidney (MDCK) cells. All of the FXYD proteins, with the exception of PLM, were primarily found in the basolateral plasma membrane. Surprisingly, PLM, a previously characterized plasma membrane protein, was found to colocalize with the ER marker protein disulfide isomerase (PDI). Treating MDCK cells expressing PLM with an agonist of PKC caused some of the PLM to be redistributed to the plasma membrane. Site-directed mutagenesis of residues within the cytoplasmic domain of PLM indicated that a negative charge at Ser-69 is necessary to shift the localization of PLM to the plasma membrane. In addition, other regions of PLM necessary for either its ER or plasma membrane localization have been elucidated. In contrast to PLM, the plasma membrane localization of CHIF and RIC was not altered by mutation of potential cytoplasmic phosphorylation sites. Overall these results demonstrate that phosphorylation of specific residues of PLM may direct PLM from an intracellular compartment to the plasma membrane.