In order to establish the role of secretagogue-induced changes in free cytosolic Ca2+ ([Ca2+]i) in pancreatic enzyme secretion, we measured the effects of carbachol, cholecystokinin-octapeptide (CCK-OP), bombesin, substance P, and bromo-A23187 on amylase release and [Ca2+]i in guinea pig pancreatic acini loaded with the Ca2+-selective fluorescent indicator, fura-2. Evaluation of time courses and dose-response curves indicated that carbachol, CCK-OP, bombesin, and substance P cause extracellular Ca2+-independent transient increases in [Ca2+]i and transient bursts in amylase release (initial secretion). The potencies for the secretagogues to increase [Ca2+]i and initial amylase release were similar. Bromo-A23187 also caused an extracellular Ca2+-independent transient increase in [Ca2+]i and amylase release. In the absence of extracellular Ca2+, sequential additions of substance P followed by carbachol caused transient increases in [Ca2+]i correlating with transient bursts in amylase release. In contrast, in acini first treated with carbachol, the ability of substance P to increase [Ca2+]i and amylase release was blocked. Sustained secretion caused by the secretagogues was dependent on extracellular Ca2+ but occurred at basal [Ca2+]i. Increasing [Ca2+]i during the sustained phase of stimulation by increasing the extracellular Ca2+ concentration or with bromo-A23187 did not increase the rate of sustained secretion.