We examined the effects of removing extracellular Ca2+ (Ca2+e), depleting intracellular Ca2+ (Ca2+i), inhibiting cAMP-dependent calmodulin, and blocking voltage-sensitive Ca2+ channels on the secretion of ACTH by perifused dispersed rat anterior pituitary cells. The cells were stimulated with synthetic arginine vasopressin (AVP), oxytocin (OT), and angiotensin-II (AII), all of which are thought to act via the Ca2+/inositol phosphate-dependent protein kinase-C pathway, with synthetic ovine CRF, which acts via the cAMP-dependent protein kinase-A pathway, and with dioctanoylglycerol, which directly activates protein kinase-C. All three secretagogues elicited an initial spike phase ACTH secretory response that peaked within 1 or 2 min and ended within 6 min. AVP and OT also elicited a sustained plateau phase response that lasted for as long as the cells were exposed to the secretagogue, but AII did not. Removal of Ca2+e diminished the initial spike phase by 30-50%, but depletion of Ca2+i virtually abolished it. In contrast, the sustained phase of the response to AVP and OT was abolished by removal of Ca2+e. The effect of dioctanoylglycerol, which elicits a sustained progressive increase in ACTH release, but no initial spike phase, was also greatly inhibited by Ca2+e removal; no greater effect was observed when Ca2+i was depleted. Blockade of L-type voltage-sensitive Ca2+ channels with nimodipine, a dihydropyridine drug, had the same effect as Ca2+e removal on both the initial spike and sustained plateau phases of the response to AVP. Inhibiting cAMP-dependent calmodulin with penfluridol had no effect on the initial spike phase, but reduced the sustained plateau phase of the response to AVP. Removal of Ca2+e or depletion of Ca2+i did not abolish the synergistic ACTH secretory response to the combination of AVP and CRF.(ABSTRACT TRUNCATED AT 400 WORDS)