Sodium pump Inhibition in cardiac muscle cells is associated with changes in intracellular sodium, calcium, and hydrogen concentrations as well as in membrane ion transport activity. We examined further the functional relations among these entities using cultured chick ventricular cells. [Ca]1 and pH, were determined from fluorescence signals obtained from cells loaded with fura-2 or BCECF, respectively. Ouabain (100 μM) elevated [Ca]1 eightfold and decreased pH, by 0.11 unit (a 30% increase in [H+]). In the presence of 10 μM ethylisopropylamiloride, a potent inhibitor of Na-H exchange, ouabain elevated [Ca]1 3.5-fold and reduced pH, by 0.16 unit (a 48% increase in [H +]). Exposure to sodium-free (sodium replaced with potassium) medium produced a twelvefold increase in [Ca], and a 0.12 pH unit decrease in pH1. In cells treated with 100 μM ouabain, exposure to sodium-free (lithium) medium resulted in a 22-fold sustained increase in [Ca], and a rapid intracellular acidification (pH 7.15 to 6.60). The effect of ouabain or sodium-free medium on pH1 was abolished in calcium-free medium; addition of 1 mM Ca rapidly increased [Ca]1 and decreased pH1. In cells treated with subtoxic (3 μM) or toxic (100 μM) concentrations of ouabain, initial 24Na uptake rates were significantly greater than in control cells and were significantly reduced in the presence of 10 μM ethylisopropylamiloride. We conclude that ouabain (100 μM) produces 1) intracellular acidification as a result of sodium pump inhibition; 2) calcium accumulation via Na-Ca exchange, and 3) subsequent Ca-H interaction within the cell. The decrease in pH, stimulates H+ efflux and Na+ influx via Na-H exchange to limit further decline in pH1 and to further augment [Na]1, which tends to elevate [Ca]1 as a result of increased calcium accumulation via Na-Ca exchange. Thus, Na-H exchange may play an important role in the positive inotropic and also the toxic effects of cardiac glycosides.