The neural mediation of acid-stimulated serotonin release was studied in isolated sheets of rabbit duodenal mucosa mounted in Ussing chambers. The serosal side of the mucosa was exposed to Ringer-HCO3 at pH 7.4, and the mucosal side was exposed to citrate-phosphate buffer at pH 3-6.8. Immunoreactive serotonin release occurred onto the luminal surface at pH 6 and below and onto the serosal surface at pH 5 and below, but was greater on the luminal side at each pH. The effect of cholinergic and adrenergic agonists and antagonists on mucosal serotonin release was measured at luminal pH 7.4, 5, and 4. Acid-stimulated luminal release was significantly inhibited by atropine, hexamethonium, and propranolol at pH 4 and 5 but not by phentolamine. Serotonin release was stimulated at pH 7.4 and 4 by carbachol and isoproterenol but not by norepinephrine or nicotine at pH 7.4. It is concluded that acid-induced and nonacid-induced mucosal serotonin release is partly neurally mediated by muscarinic cholinergic and beta-adrenergic mechanisms.