The Na,K-ATPase α₂ subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca²⁺, 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 α₂β₂ heterodimers. We have expressed human α₁β₁, α₂β₁, α₂β₂, and α₂β₃ in Pichia pastoris, purified the complexes, and compared their functional properties. α₂β₂ and α₂β₃ differ significantly from both α₂β₁ and α₁β₁ in having a higher K_0.5K⁺ and lower K_0.5Na⁺ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K⁺ and shift of the E₁P-E₂P conformational equilibrium toward E₁P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α₂β₂ and α₂β₃ over α₁β₁ (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 α₂β₂ could allow an acute response to raised ambient K⁺ concentrations in physiological conditions and explain the importance of α₂β₂ for cardiac muscle contractility. The high sensitivity of α₂β₂ to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α₂β₂-selective digoxin derivatives for reducing cardiotoxicity.