Incubating dispersed acini from guinea pig pancreas with cholecystokinin and then washing the cells to remove cholecystokinin reduced the subsequent stimulation of amylase secretion caused by pancreatic secretagogues, whose actions are mediated by release of cellular calcium (i.e., cholecystokinin, carbamylcholine, bombesin, litorin, physalaemin, and A23187), but did not alter the stimulation caused by secretagogues whose actions are mediated by cAMP (i.e., vasoactive intestinal peptide and secretin). This cholecystokinin-induced desensitization was reversible, and the onset of the process as well as its reversal were time- and temperature-dependent changes. The concentrations of cholecystokinin required to cause desensitization were greater than those required to cause maximal stimulation of amylase secretion, and this finding suggests that the submaximal stimulation of enzyme secretion seen with supramaximal concentrations of cholecystokinin may be caused by cholecystokinin-induced desensitization.