When dispersed chief cells from guinea pig stomach were first incubated with cholecystokinin (CCK), washed, and then reincubated with CCK in fresh incubation solution, the stimulation of pepsinogen secretion and the rise in intracellular calcium concentration during the second incubation were reduced. CCK did not cause residual enzyme secretion but caused desensitization that was rapid, temperature dependent, dependent on extracellular calcium, reversible with time, and prevented but not reversed by CCK receptor antagonists. Cholecystokinin octapeptide (CCK-8) caused desensitization over the same range of concentrations that stimulate pepsinogen secretion, whereas the concentration of gastrin required to cause maximal desensitization was greater than that required to cause maximal stimulation of enzyme secretion. CCK-8 caused heterologous desensitization of pepsinogen secretion stimulated by agonists that interact with receptors to cause mobilization of cellular calcium and activation of protein kinase C or by agonists that bypass receptors to activate these mediators directly; however, CCK-8 did not induce desensitization of the stimulation caused by any secretagogue whose actions are mediated by adenosine 3',5'-cyclic monophosphate. Because CCK-8 caused greater desensitization of secretion stimulated by agonists that interact with receptors than by agonists that bypass receptors, it is likely that receptor modulation as well as a postreceptor action contribute to the ability of CCK to cause desensitization of pepsinogen secretion from chief cells.