In dispersed guinea pig pancreatic acini, bombesin stimulates a rapid increase in the concentration of free cytosolic Ca2+ ([Ca2+]i) by releasing Ca2+ from an intracellular agonist-sensitive pool. This increase is transient, and it mediates the initial secretion of digestive enzymes from the acinar cell. During sustained bombesin stimulation, [Ca2+]i returns to the resting or near-resting level, but bombesin-stimulated enzyme secretion continues. Although [Ca2+]i is at the resting level during continued stimulation, 45Ca2+ flux measurements indicate that bombesin continues to release Ca2+ from an intracellular store that is probably the agonist-sensitive pool. Low concentrations (0.3 microM) of the non-fluorescent Ca2+ ionophore, 4-bromo-A23187 (Br-A23187), release Ca2+ from the agonist-sensitive pool without measurably increasing [Ca2+]i. This ionophore-induced Ca2+ release without production of an increase in [Ca2+]i does not by itself stimulate enzyme secretion, but it does augment the secretion caused by the phorbol ester, 4 beta-phorbol, 12-myristate, 13-acetate (PMA). These results suggest a role for intracellular Ca2+ release in bombesin-stimulated sustained secretion.