The responsiveness of anterior pituitary tumor (GH3) cells to promoters of prolactin secretion and/or synthesis and cyclic AMP accumulation was studied as a function of cellular Ca2+ content. GH3 cells exposed to media containing 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid were reduced 7-fold in Ca2+ content without loss of viability. Preparations of Ca2+-depleted cells were largely unchanged in cyclic AMP content when challenged by thyrotropin-releasing hormone (TRH), whereas cells which were subsequently restored at optimal Ca2+ (0.5 mM) responded to the hormone with 2- to 3-fold increases in cyclic AMP content. The decreased responsiveness of Ca2+-depleted cells to TRH was not influenced by phosphodiesterase inhibitors, incubation time, or hormone concentration. TRH-dependent cyclic AMP accumulation was markedly potentiated by forskolin in Ca2+-restored, but not in Ca2+-depleted, cell preparations. Forskolin extended the time period during which cyclic AMP accumulated in response to TRH without altering the TRH concentration dependency of the cells. Varying increases in GH3 cyclic AMP content occurred in response to other hormones or agents which enhance prolactin secretion and/or synthesis. In Ca2+-restored cells, cyclic AMP content was increased 2-fold by prostaglandin E1 (PGE1) and epidermal growth factor (EGF), 10- to 15-fold by vasoactive intestinal polypeptide (VIP) and 6-fold by phorbol myristate acetate (PMA); the capacity of Ca2+-depleted cells, however, to accumulate cyclic AMP in response to PGE1, EGF, and VIP was greatly reduced. Accumulation of cyclic AMP following short-term incubations with cholera toxin similarly was dependent on Ca2+. Exposure of GH3 cells preloaded with 45Ca to TRH, PGE1, EGF, PMA, or VIP resulted in losses of cell-associated 45Ca. Pretreatment with these agents resulted in a decreased capacity of the cells to accumulate 45Ca from the extracellular medium. The results of this study support the hypothesis that various putative humoral regulators of prolactin secretion and/or synthesis act on GH3 cells to alter intracellular Ca2+ metabolism which in turn results in an increased cyclic AMP content through stimulation of adenylate cyclase activity.