To catalyze ion transport, the Na,K-ATPase must contain one α and one β subunit. When expressed by transfection in various expression systems, each of the four α subunit isoforms can assemble with each of the three β subunit isoforms and form an active enzyme, suggesting the absence of selective α-β isoform assembly. However, it is unknown whether in vivo conditions the α-β assembly is random or isoform-specific. The α(2)-β(2) complex was selectively immunoprecipitated by both anti-α(2) and anti-β(2) antibodies from extracts of mouse brain, which contains cells co-expressing multiple Na,K-ATPase isoforms. Neither α(1)-β(2) nor α(2)-β(1) complexes were detected in the immunoprecipitates. Furthermore, in MDCK cells co-expressing α(1), β(1), and β(2) isoforms, a greater fraction of the β(2) subunits was unassembled with α(1) as compared with that of the β(1) subunits, indicating preferential association of the α(1) isoform with the β(1) isoform. In addition, the α(1)-β(2) complex was less resistant to various detergents than the α(1)-β(1) complex isolated from MDCK cells or the α(2)-β(2) complex isolated from mouse brain. Therefore, the diversity of the α-β Na,K-ATPase heterodimers in vivo is determined not only by cell-specific co-expression of particular isoforms, but also by selective association of the α and β subunit isoforms.