The carboxy terminus (CT) of the colonic H(+)-K(+)-ATPase is required for stable assembly with the beta-subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HKalpha(2), we selected 84 amino acids in the CT of the alpha-subunit of mouse colonic H(+)-K(+)-ATPase (CT-HKalpha(2)) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HKalpha(2) with CD63, recombinant CT-HKalpha(2) and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HKalpha(2) protein (but not CT-HKalpha(1)) coprecipitated with CD63, confirming stable assembly of HKalpha(2) with CD63. In HEK-293 transfected with HKalpha(2) plus beta(1)-Na(+)-K(+)-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKalpha(2)/NKbeta(1) and (86)Rb(+) uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKalpha(2)-NKbeta(1) complex in the cell membrane.