Muscarinic acetylcholine receptors play an important role in the regulation of gastric acid secretion stimulated by acetylcholine; nonetheless, the precise role of each receptor subtype (M1–M5) remains unclear. This study examined the involvement of M1, M3, and M5 receptors in cholinergic regulation of acid secretion using muscarinic receptor knockout (KO) mice. Gastric acid secretion was measured in both mice subjected to acute gastric fistula production under urethane anesthesia and conscious mice that had previously undergone pylorus ligation. M3 KO mice exhibited impaired gastric acid secretion in response to carbachol. Unexpectedly, M1 KO mice exhibited normal intragastric pH, serum gastrin and mucosal histamine levels, and gastric acid secretion stimulatied by carbachol, histamine, and gastrin. Pirenzepine, known as an M1-receptor antagonist, inhibited carbachol-stimulated gastric acid secretion in a dose-dependent manner in M1 KO mice as well as in wild-type (WT) mice, suggesting that the inhibitory effect of pirenzepine on gastric acid secretion is independent of M1-receptor antagonism. Notably, M5 KO mice exhibited both significantly lower carbachol-stimulated gastric acid secretion and histamine-secretory responses to carbachol compared with WT mice. RT-PCR analysis revealed M5-mRNA expression in the stomach, but not in either the fundic or antral mucosa. Consequently, cholinergic stimulation of gastric acid secretion is clearly mediated by M3 (on parietal cells) and M5 receptors (conceivably in the submucosal plexus), but not M1 receptors.