Analysis of the role of calcium in the regulation of aldosterone production showed that the steroidogenic responses of isolated adrenal cells to ACTH, angiotensin II, and potassium were highly dependent on the extracellular calcium concentration. Physiological concentrations of calcium were required for maximal aldosterone responses to all three regulators, and steroidogenesis was progressively reduced by decreasing calcium concentrations. ACTH stimulated aldosterone and cAMP responses at all calcium concentrations tested, and the production of steroid was correlated with the increase in cAMP formation. Reduction of extracellular calcium concentration caused an increase in the ACTH concentration required for halfmaximal steroid and cAMP production and a decline in the maximal output of aldosterone. When zona glomerulosa cells were stimulated by exogenous cAMP, cholera toxin, or serotonin, decreased calcium concentration caused a decline in the amount of steroid produced but did not change the agonist concentration required for half-maximal stimulation. These findings suggest that calcium is required for the coupling of ACTH-receptor complexes with adenylate cyclase in the glomerulosa cell membrane and also at an intracellular locus subsequent to the action of cAMP. In contrast, angiotensin II and potassium did not increase cAMP production at any of the calcium concentrations tested, but both regulators stimulated aldosterone production in a calcium-dependent manner. Reduction of extracellular calcium concentration caused a decrease in the amount of steroid produced but did not change the concentration of either angiotensin II or potassium required for half-maximal stimulation of aldosterone production. Binding of angiotensin II to glomerulosa cell receptors was not altered in the absence of calcium, again indicating the postreceptor locus of a calcium-dependent step in the steroidogenic pathway. Although calcium is required for the steroidogenic response of zona glomerulosa cells to ACTH, angiotensin II, and potassium, the calcium requirement for ACTH action is quantitatively distinct from that for angiotensin II and potassium, consistent with a more calcium-dependent mechanism of action of the latter regulators.