It has been demonstrated that ascorbic acid is present in human gastric juice at a very high level even in the fasting state. In this study, we confirmed such a physiological event in pylorus-ligated rats and investigated the mechanism for gastric ascorbic acid secretion using the rat in vivo perfusion method. Gastric juice from fasting rats after a 4-hr ligation contained a 6-fold higher level of ascorbic acid than that in sera, indicating the presence of its active transport system across the gastric wall. To identify an endogenous mediator, four kinds of gastric stimulants were compared for their secretory abilities. Although all four secretagogues used stimulated acid secretion to comparable levels, ascorbic acid secretion was stimulated only by carbamylcholine chloride (carbachol). Carbachol-stimulated ascorbic acid secretion was abolished by atropine pretreatment, showing that it is mediated via a muscarinic cholinergic receptor. This cholinergic stimulation was also demonstrated in a rat mutant that genetically lacks the ability to synthesize ascorbic acid in the liver, similar to humans. In addition, a potent inhibitor of the gastric proton pump, which is a final regulatory component in the mechanism of acid secretion, caused no inhibition of ascorbic acid secretion in Osteogenic Disorder Shionogi (ODS) rats. These results are the first evidence indicating that ascorbic acid, the reduced form of vitamin C, is actively and independently secreted into the gastric juice under regulation of a cholinergic receptor system.