Uroguanylin and guanylin are endogenous ligands for guanylate cyclase C, an upstream regulator of the cystic fibrosis transmembrane resistance (CFTR) anion secretory channel. Present evidence suggests that uroguanylin is produced by enteroendocrine and guanylin by goblet cells in the small intestinal epithelium. These cell types may act as sensory cells, registering luminal contents and initiating proper intestinal responses. Our aim was to elucidate possible interactions between guanylins and some other stimuli of intestinal secretion, including melatonin. Methods: Male Lewis x Dark Agouti rats were anesthetized and a 12-mm segment of proximal duodenum with intact blood supply was cannulated in situ. Mucosal bicarbonate secretion (pH stat) and mean arterial blood pressure were continuously recorded. Guanylins were administered by close intra-arterial infusion or added to the luminal perfusate. Results: Intra-arterial infusion (50-1000 pmol/kg,h) as well as luminal administration (0.05-0.5 mM) of both guanylin and uroguanylin caused dose-dependant increases in duodenal bicarbonate secretion. Responses were considerably larger after luminal administration. The muscarinic antagonist atropine (0.5 mg/kg bolus + 0.5 mg/kg,h, i.v.) completely abolished the stimulatory action of intra-arterial uroguanylin. The melatonin MT₂-selective antagonist luzindole significantly depressed secretory responses to intra-arterial guanylins but did not affect responses to luminal administration of the peptides. Conclusions: Guanylins, primarily uroguanylin, are potent stimuli of duodenal electrolyte secretion. The secretory response to basolaterally administered uroguanylin seems dependant on intact muscarinic signalling and influenced by the neurohormone melatonin.