Intracellular ionized Ca2+ concentration was measured in clonal mouse anterior pituitary tumor cells with the fluorescent Ca2+ indicator Quin-2. In control physiological solution, free cytoplasmic Ca2+ concentration was found to be 139 +/- 11 nM. Replacement of 50 mM NaCl by 50 mM KCl in the extracellular fluid caused a 29 mV depolarization and a 4.2-fold increase in the concentration of free cytoplasmic Ca2+. Under comparable depolarizing conditions, a specific influx of 2.66 nmole of 45Ca2+ per mg protein was detected 1 min after addition of high K+, accompanied by a marked increase in the initial rate of beta-endorphin secretion. In the absence of external Ca2+, depolarization by K+ produced little or no increase in either intracellular free Ca2+ or hormone release. Incubation of AtT-20/D16-16 cells in the secretagogue norepinephrine led to a depolarization accompanied by an increase in spontaneous action potential frequency and a marked elevation in cytosolic Ca2+ concentration. Exposure of cells to somatostatin, an inhibitor of hormone release, led to only transient decreases in burst frequency and no significant reduction in intracellular Ca2+ levels. These results indicate that in addition to intracellular Ca2+, other factors also control secretory activity in AtT-20/D16-16 anterior pituitary cells.