Cardiac cells express more than one isoform of the Na, K-ATPase (NKA), the heteromeric enzyme that creates the Na(+) and K(+) gradients across the plasmalemma. Cardiac isozymes contain one catalytic ?-subunit isoform (?1, ?2, or ?3) associated with an auxiliary ?-subunit isoform (?1 or ?2). Past studies using biochemical approaches have revealed minor kinetic differences between isozymes formed by different ?-? isoform combinations; these results make it difficult to understand the physiological requirement for multiple isoforms. In intact cells, however, NKA enzymes operate in a more complex environment, which includes a substantial transmembrane potential. We evaluated the voltage dependence of human cardiac NKA isozymes expressed in Xenopus oocytes, and of native NKA isozymes in rat ventricular myocytes, using normal mammalian physiological concentrations of Na(+)o and K(+)o. We demonstrate that although ?1 and ?3 pumps are functional at all physiologically relevant voltages, ?2?1 pumps and ?2?2 pumps are inhibited by ?75% and ?95%, respectively, at resting membrane potentials, and only activate appreciably upon depolarization. Furthermore, phospholemman (FXYD1) inhibits pump function without significantly altering the pump's voltage dependence. Our observations provide a simple explanation for the physiological relevance of the ?2 subunit (?20% of total ? subunits in rat ventricle): they act as a reserve and are recruited into action for extra pumping during the long-lasting cardiac action potential, where most of the Na(+) entry occurs. This strong voltage dependence of ?2 pumps also helps explain how cardiotonic steroids, which block NKA pumps, can be a beneficial treatment for heart failure: by only inhibiting the ?2 pumps, they selectively reduce NKA activity during the cardiac action potential, leading to an increase in systolic Ca(2+), due to reduced extrusion through the Na/Ca exchanger, without affecting resting Na(+) and Ca(2+) concentrations.