The assembly of the -subunit of the gastric H-K-ATPase (HK) with the -subunit of the H-K-ATPase or the Na-K-ATPase (NaK) was characterized with two anti-HK monoclonal antibodies (MAbs). In fixed gastric oxyntic cells, in H-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cells transfected with HK, MAb 2/2E6 was observed to bind to HK only when interactions between - and -subunits were disrupted by various denaturants. The epitope for MAb 2/2E6 was mapped to the tetrapeptide S₂₂₆LHY₂₂₉ of the extracellular domain of HK. The epitope for MAb 2G11 was mapped to the eight NH₂-terminal amino acids of the cytoplasmic domain of HK. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HK with -subunits of the endogenous cell surface NaK, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HK. In HK-transfected LLC-PK₁ cells, significant immunofluorescent labeling of HK at the cell surface could be detected without postfixation denaturation or in live cells, although a fraction of transfected HK could also be coimmunoprecipitated with NaK. Thus assembly of HK with NaK does not appear to be a stringent requirement for cell surface delivery of HK in LLC-PK₁ cells but may be required in MDCK cells. In addition, endogenous posttranslational regulatory mechanisms to prevent hybrid - heterodimer assembly appear to be compromised in transfected cultured renal epithelial cells. Finally, the extracellular epitope for assembly-sensitive MAb 2/2E6 may represent a region of HK that is associated with - interaction.