In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the, Na,K-ATPase and subunits led us to question whether this was alterable and thus if some form of regulation was involved. We have examined the consequences of over-expressing Na, K-ATPase ₁ subunits using MDCK cells expressing flag-tagged ₁ subunits (₁flag) or myc -tagged ₁ subunits (₁myc) under the control of a tetracycline-dependent promoter. The induction of ₁flag subunit synthesis in MDCK cells, which increases ₁-subunit expression at the plasma membrane by more than 2-fold while maintaining stable 1 expression levels, revealed that all mature ₁ subunits associate with ₁ subunits and no evidence of "free" ₁ subunits was obtained. Consequently, the ratio of assembled ₁/₁ subunits is significantly increased when "extra" subunits are expressed. An increased ₁/₁ stoicheometry is also observed in cells treated with tunicamycin, suggesting that the protein:protein interactions involved in these complexes are not dependent on glycosylation. Confocal images of co-cultured ₁myc-expressing and ₁flag-expressing MDCK cells show colocalization of ₁myc and ₁flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the subunits. Immunoprecipitation using MDCK cells constitutively expressing ₁myc and tetracycline-regulated ₁flag subunits confirmed - subunit interactions. These results demonstrate that the equimolar ratio of assembled ₁/₁ subunits of the Na,K-ATPase in kidney cells is not fixed by the inherent properties of the interacting subunits. It is likely that cellular mechanisms are present that regulate the individual Na,K-ATPase subunit abundance.