The carboxy terminus (CT) of the colonic H+-K+-ATPase is required for stable assembly with the β-subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HKα2, we selected 84 amino acids in the CT of the α-subunit of mouse colonic H+-K+-ATPase (CT-HKα2) 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-HKα2 with CD63, recombinant CT-HKα2 and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HKα2 protein (but not CT-HKα1) coprecipitated with CD63, confirming stable assembly of HKα2 with CD63. In HEK-293 transfected with HKα2 plus β1-Na+-K+-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKα2/NKβ1 and 86Rb+ uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKα2-NKβ1 complex in the cell membrane.