Background and aim: Melatonin is released from enterochromaffin cells in the intestinal mucosa and from the pineal gland in the CNS, but its influence on gastrointestinal function is largely unknown. The aim of the present study was to elucidate the effects of melatonin on duodenal motility, mucosal permeability, net fluid flux and duodenal bicarbonate secretion (DBS). Furthermore, we also studied the effects of melatonin on ethanol-induced changes of intestinal barrier function. Methods: Rats were anaesthetized with thiobarbiturate and a ~30 mm segment of the proximal duodenum with an intact blood supply was perfused in situ with saline or saline containing either melatonin or ethanol. Effects on duodenal motility, mucosal permeability [blood-to-lumen clearance of 51Cr-EDTA], net fluid flux and DBS were investigated. Agents were administered luminally and/or intravenously (iv). Results: 50 M of luminal melatonin significantly increased DBS, reduced mucosal permeability, increased duodenal motility. Luminal perfusion with 10% ethanol induced ~two-fold increases in both DBS and mucosal permeability, as well as a transient increase in duodenal motility. 15% ethanol caused a ~three-fold increase in DBS, a ~ten-fold increase in mucosal permeability and an immediate and sustained increase duodenal motility. Pre-treating the animals with melatonin 20 mg/kg iv strongly reduced the response to ethanol. Hexamethonium (10 mg/kg iv followed by continuous iv infusion at a rate of 10 mg/kg/h), abolished the effects of iv melatonin. Conclusions: Melatonin significantly decreases basal duodenal mucosal permeability and increases DBS. Melatonin also reduces ethanol-induced increases in mucosal permeability and motility, probably via enteric neural pathways involving nicotinic receptors.