The role of calcium in the stimulation of ACTH secretion by CRF and other regulators was studied in rat anterior pituitary cells. Incubation of cultured pituitary cells in normal calcium with CRF, vasopressin, angiotensin II, or norepinephrine increased the rate of ACTH release for up to 45 min and then became constant for up to 3 h. In the absence of extracellular calcium, the initial rate of stimulated secretion was unaffected, but after 45 min the secretion rate decreased by 40% for CRF and to a greater extent for the other stimuli. Addition of calcium after 90 min in calcium-free medium restored the CRF-stimulated ACTH release rate to the control value. The absence of extracellular calcium had no effect on CRF-stimulated cAMP accumulation, but intracellular calcium depletion by preincubation of the cells with EGTA completely inhibited CRF-stimulated cAMP production and ACTH release. The voltage-dependent calcium channel antagonist nitrendipine and the calcium channel agonist BK 8644 had little effect on the CRF-stimulated ACTH release rate, while they, respectively, inhibited and enhanced the stimulation by vasopressin and high potassium. In calcium-depleted cells incubated with the calcium ionophore A23187, CRF stimulation of cAMP production and ACTH release were dependent upon extracellular calcium concentrations from 0.1-100 microM. These findings have defined two phases in the stimulation of ACTH release by CRF and cAMP-independent stimuli in cultured pituitary cells: an early phase with a rapid increase in the ACTH release rate which is independent of extracellular calcium, and a late phase of constant secretion rate, with partial extracellular calcium dependence for the stimulation by CRF and complete calcium dependence for the stimulation by non-cAMP-mediated stimuli.