The Na+/K+ ATPase is composed of two subunits called alpha and beta chains. In insect cells, independently expressed alpha and beta chains are localized to intracellular membranes. Sucrose density gradient sedimentation, crosslinking analysis, and immunoprecipitation of radio-labeled proteins show that the alpha chains expressed alone are in large aggregates of different molecular weights with less than 4% being monomeric. Analysis by non-reducing SDS-PAGE and immunoblotting show that the beta chains expressed alone are in Triton X-100 insoluble, disulfide-linked aggregates. Co-expression of both subunits in insect cells results in only a small fraction (less than 15%) of the alpha chains being assembled as the active recombinant enzyme, with at least 22% of the active recombinant enzyme localized to the plasma membrane as determined by a biochemical assay. The small amount of beta chain at the plasma membrane in cells that express both subunits is beyond the limit of detection by the biochemical assay. Immunoprecipitation of Triton X-100 soluble alpha chains from radio-labeled cells expressing both subunits shows that the alpha chains are mostly in large aggregates containing beta chains. These results suggest that, in insect cells, the availability of correctly folded beta chains is the rate limiting step in the assembly of active Na+/K+ ATPase.