Mat-8 was fused with a Myc-tag or green fluorescent protein at its carboxyl terminus, and then expressed in Chinese hamster ovary K1 cells. Determination of the cellular localization of the tagged proteins suggested that they were localized on the intracellular membrane, being not only detected around the nuclear envelope but also partly overlapping with markers for endosomes and Golgi bodies. However, Mat-8 with the Myc-tag was detected on the plasma membrane as well as the intracellular membrane, when it was expressed in colorectal cancer cells. The membrane fraction of the cancer cells was solubilized and immuno-precipitated with an antibody for the Myc-tag. Western-blotting analysis demonstrated that the Na+/K+-ATPase alpha subunit was present in the precipitate. Furthermore, the immuno-precipitate obtained with an antibody for the Na+/K+-ATPase alpha subunit reacted with that for the Myc-tag. These results suggested that Mat-8 could be associated with Na+/K+-ATPase similar to other FXYD family members. The Gly41-->Arg mutation in the transmembrane region of Mat-8 inhibited its association with the Na+/K+-ATPase alpha subunit and localization on the plasma membrane, whereas the Cys44-->Ala or Cys49-->Ala substitution did not. Thus the conserved Gly41 residue in the transmembrane domain could be indispensable for localization of Mat-8 on the cell surface.