Because the activity of the sodium pump (Na-K-ATPase) influences the secretion of aldosterone, we determined how extracellular potassium (K_o) and calcium affect sodium pump activity in rat adrenal glomerulosa cells. Sodium pump activity was measured as ouabain-sensitive ⁸⁶Rb uptake in freshly dispersed cells containing 20 mM sodium as measured with sodium-binding benzofluran isophthalate. Increasing K_o from 4 to 10 mM in the presence of 1.8 mM extracellular calcium (Ca_o) stimulated sodium pump activity up to 165% and increased intracellular free calcium as measured with fura 2. Increasing K_o from 4 to 10 mM in the absence of Ca_o stimulated the sodium pump ~30% and did not increase intracellular free calcium concentration ([Ca²⁺]_i). In some experiments, addition of 1.8 mM Ca_o in the presence of 4 mM K_o increased [Ca²⁺]_i above the levels observed in the absence of Ca_o and stimulated the sodium pump up to 100%. Ca-dependent stimulation of the sodium pump by K_o and Ca_o was inhibited by isradipine (10 µM), a blocker of L- and T-type calcium channels, by compound 48/80 (40 µg/ml) and calmidizolium (10 µM), which inhibits calmodulin (CaM), and by KN-62 (10 µM), which blocks some forms of Ca/CaM kinase II (CaMKII). Staurosporine (1 µM), which effectively blocks most forms of protein kinase C, had no effect. In the presence of A-23187, a calcium ionophore, the addition of 0.1 mM Ca_o increased [Ca²⁺]_i to the level observed in the presence of 10 mM K_o and 1.8 mM Ca_o and stimulated the sodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mM Ca_o was not reduced by isradipine but was blocked by KN-62. Thus, under the conditions that K_o stimulates aldosterone secretion, it stimulates the sodium pump by two mechanisms: direct binding to the pump and by increasing calcium influx, which is dependent on Ca_o. The resulting increase in [Ca²⁺]_i may stimulate the sodium pump by activating CaM and/or CaMKII.