AbstractWhen dispersed chief cells from guinea pig stomach were first incubated with carbachol, washed, and then reincubated with carbachol in fresh incubation solution, the stimulation of pepsinogen secretion and the rise in intracellular calcium concentration during the second incubation were reduced. Carbachol did not cause residual enzyme secretion, but the same range of concentrations that causes enzyme secretion caused desensitization that was rapid, temperature dependent, and reversible with time. Preincubation with carbachol caused approximately a 65% reduction in enzyme secretion stimulated during a subsequent incubation with this agonist, but the potency of carbachol was unaffected. Prior exposure to carbachol also reduced subsequent stimulation caused by cholecystokinin (CCK‐8), gastrin I, ionophore A23187, or 12‐O‐tetradecanoylphorbol 13‐acetate but did not alter stimulation by any agonist that increases cellular cAMP. Carbachol pretreatment of Fura‐loaded chief cells caused a threefold increase in the EC50for carbachol‐stimulated [Ca2+]iand approximately a 30% reduction in the maximal rise in [Ca2+]iin response to carbachol or CCK‐8. Inhibition of [N‐methyl‐3H] scopolamine binding by carbachol following carbachol pretreatment indicated that modulation of receptor affinity or number did not account for functional desensitization. These data indicate that carbachol causes heterologous desensitization of pepsinogen secretion stimulated by agonists that mobilize cellular Ca2+or activate protein kinase C through a postreceptor action and suggest that an attenuated rise in chief cell calcium is one mechanism mediating the desensitization of