The release of immunoreactive ACTH (IR-ACTH) from AtT-20 pituitary tumor cells was transiently increased by exposure to an elevated concentration of potassium ion in an osmotically balanced extracellular medium. With the calcium-sensitive dye Quin 2, the concentration of free cytosolic calcium (Cai) in the AtT-20 tumor was determined to be 115 nM. Challenge of these cells with 60 mM potassium in an osmotically balanced salt solution raised the concentration of Cai to 246 nM. This is in accord with the view that agents promoting calcium entry into pituitary cells trigger hormone secretion. Addition of forskolin to the extracellular medium caused a sustained release of IR-ACTH from AtT-20 tumor cells. Challenge with forskolin (10 microM) increased the concentration of Cai to 149 nM. This observation is also in accord with the view that calcium entry is a necessary and sufficient stimulus to trigger hormone secretion from the anterior pituitary lobe. Exposure of cells to forskolin (10 microM) before a potassium challenge increased the quantity of IR-ACTH released in response to potassium, but did not alter the minute by minute time course of the response to this ion. Forskolin pretreatment did not alter the potassium-evoked rise in Cai concentration. This observation suggests that the magnitude of the secretory response of the pituitary gland can be enhanced by agents other than those promoting an increase in Cai. After exposure of the tumor cells to potassium for a sufficient time to permit the rate of release of hormone to return to the basal value, forskolin could still stimulate the release of hormone from the tumor cells. Under these circumstances, forskolin did not increase the concentration of Cai. This observation suggests that pituitary hormone secretion can be initiated by a factor(s) other than an acute change in the Cai concentration. Both forskolin and 8-bromo-cAMP stimulated hormone secretion from dispersed melanotrophs and potentiated the potassium-evoked secretory response of these cells. Neither compound affected the apparent time course of the response to potassium. These observations suggest that the effects of forskolin and potassium on the AtT-20 tumor cell may use mechanisms occurring in normal pituitary cells.