We have found that immortalized aortic myocytes have high Na(+)-Ca2+ exchange activity that is similar in amount to that of low-passage myocyte cultures. Replacing all external Na+ with K+ slightly increased 45Ca2+ uptake in cells with basal Na+ and greatly increased 45Ca2+ uptake in cells with increased intracellular Na+. External Na+ competitively inhibited 45Ca2+ uptake (inhibition constant approximately 10 mM). The dependence of exchange activity on intracellular Na+ was sigmoidal. The 50% maximum concentration (K0.5) for Na+ was 33 +/- 1 mmol/l cell water space. The Hill coefficient was 2.9 +/- 0.2, which is consistent with a stoichiometry of 3 Na+/1 Ca2+. Cytosolic free Na+ was estimated from the ratio of sodium-binding benzofuran isophthalate (SBFI) fluorescence and an in vivo calibration curve. Sulfinpyrazone, an inhibitor of anion transport, markedly increased the SBFI content of the cells without affecting cell Na+. Cytosolic free Na+ was 9 mM under basal conditions. Because free Na+ did not differ significantly from total Na+, little or no Na+ is bound or compartmentalized in these cells. The complete replacement of extracellular Na+ with K+ fails to evoke appreciable Ca2+ influx, at least in part, because of the low cell Na+ concentration relative to the K0.5 of the exchanger and its sigmoid dependence on Na+.