By participating in the regulation of ion and voltage gradients, the Na-K pump (i.e., Na,K-ATPase) influences many aspects of cellular physiology. Of the four alpha isoforms of the pump, alpha1 is ubiquitous, alpha2 is predominant in skeletal muscle, and alpha3 is found in neurons and the cardiac conduction system. To determine whether the isoforms have different intracellular Na+ affinities, we used the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI) to measure pump-mediated Na+ efflux as a function of [Na+]i in human HeLa cells stably transfected with rat Na-K pump isoforms. We Na+-loaded the cells, and then monitored the time course of the decrease in [Na+]i after removing external Na+. All transfected rat alpha subunits were highly ouabain resistant: the alpha1 isoform is naturally resistant, whereas the alpha2 and alpha3 isoforms had been mutagenized to render them resistant. Thus, the Na+ efflux mediated by endogenous and transfected pumps could be separated by studying the cells at low (1 microM) and high (4 mM) ouabain concentrations. We found that the apparent Km for Na+ efflux attributable to the native human alpha1 isoform was 12 mM, which was similar to the Km of rat alpha1. The alpha2 and alpha3 isoforms had apparent Km's of 22 and 33 mM, respectively. The cells expressing alpha3 had a high resting [Na+]i. The maximal activity of native alpha1 in the alpha3-transfected cells was only approximately 56% of native alpha1 activity in untransfected HeLa cells, suggesting that transfection with alpha3 led to a compensatory decrease in endogenous alpha1 pumps. We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence alpha1 < alpha2 < alpha3. The alpha3 isoform may be suited for handling large Na+ loads in electrically active cells.