The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca 2+ , whereas α1 has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α- β heterodimers. It is not known whether the distinct role of α2 in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with β2 in T-tubules of cardiac myocytes, forming α2 β2 heterodimers. We have expressed human α1 β1 , α2 β1 , α2 β2 , and α2 β3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2 β2 and α2 β3 differ significantly from both α2 β1 and α1 β1 in having a higher K0.5 K+ and lower K0.5 Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1 P-E2 P conformational equilibrium toward E1 P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2 β2 and α2 β3 over α1 β1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2 β2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2 β2 for cardiac muscle contractility. The high sensitivity of α2 β2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2 β2 -selective digoxin derivatives for reducing cardiotoxicity.