FXYD5 is a member of a family of tissue specific regulators of the Na⁺/K⁺-ATPase expressed in kidney tubules. Previously we have shown that FXYD5 interacts with the subunits of the Na⁺/K⁺-ATPase and increases its V_max. The current study further characterizes structural interaction and structure-function relationships of FXYD5. FXYD5/FXYD4 chimera expressed in Xenopus oocytes have been used to demonstrate that both the high affinity association with the pump and the increase in V_max are mediated by the transmembrane domain of FXYD5. Several amino acids that participate in the high affinity interaction between FXYD5 and the subunit of the Na⁺/K⁺-ATPase have been identified. The data suggest that different FXYD proteins interact similarly with the Na⁺/K⁺-ATPase and their transmembrane domains play a key role in both the structural interactions and functional effects. Other experiments have identified at least one splice variant of FXYD5 with 10 additional amino acids at the C-terminus, suggesting the possibility of other functional effects not mediated by the transmembrane domain. FXYD5 could be specifically bound to wheat germ agglutinin beads indicating that it is glycosylated. However, unlike previous findings in metastatic cells, such glycosylation does not evoke a large increase in the size of the protein expressed in native epithelia and Xenopus oocytes.