Muscarinic cholinergic mechanisms play a key role in stimulating gastric pepsinogen secretion. Studies using antagonists suggested that the M₃ receptor subtype (M₃R) plays a prominent role in mediating pepsinogen secretion, but in situ hybridization indicated expression of M₁ receptor (M₁R) in rat chief cells. We used mice that were deficient in either the M₁ (M₁R^−/−) or M₃ (M₃R^−/−) receptor or that lacked both receptors (M_1/3R^−/−) to determine the role of M₁R and M₃R in mediating cholinergic agonist-induced pepsinogen secretion. Pepsinogen secretion from murine gastric glands was determined by adapting methods used for rabbit and rat stomach. In wild-type (WT) mice, maximal concentrations of carbachol and CCK caused a 3.0- and 2.5-fold increase in pepsinogen secretion, respectively. Maximal carbachol-induced secretion from M₁R^−/− mouse gastric glands was decreased by 25%. In contrast, there was only a slight decrease in carbachol potency and no change in efficacy when comparing M₃R^−/− with WT glands. To explore the possibility that both M₁R and M₃R are involved in carbachol-mediated pepsinogen secretion, we examined secretion from glands prepared from M_1/3R^−/− double-knockout mice. Strikingly, carbachol-induced pepsinogen secretion was nearly abolished in glands from M_1/3R^−/− mice, whereas CCK-induced secretion was not altered. In situ hybridization for murine M₁R and M₃R mRNA in gastric mucosa from WT mice revealed abundant signals for both receptor subtypes in the cytoplasm of chief cells. These data clearly indicate that, in gastric chief cells, a mixture of M₁ and M₃ receptors mediates cholinergic stimulation of pepsinogen secretion and that no other muscarinic receptor subtypes are involved in this activity. The development of a murine secretory model facilitates use of transgenic mice to investigate the regulation of pepsinogen secretion.