It is presently unknown whether Ca 2+ plays a role in the physiological control of Na +/K +-ATPAse or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca 2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca 2+ on the sodium pump were examined with studies of [ 3H]ouabain binding and 86Rb + uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K +, a reduction of the extracellular Ca 2+ increased specific [ 3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca 2+ was lowered to about 50 μM. The change in [ 3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb + uptake was increased by a reduction of extracellular Ca 2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na + influx failed to influence the inhibitory effect of Ca 2+ on glycoside binding. When [ 3H]ouabain binding was at equilibrium, the addition of Ca 2+ decreased and that of EGTA increased the glycoside binding. Mn 2+, which does not penetrate the cell membrane, had effects similar to Ca 2+. In the absence of K +, cells lose their tolerance to Ca 2+. Reducing Ca 2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [ 3H]ouabain binding observed in the absence of K +. These results indicate that a large change in extracellular Ca 2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.