Melatonin and its synthesizing enzymes have been demonstrated in the gastrointestinal tract, suggesting the in situ biosynthesis of melatonin by the gut tissues. There appears to be a diurnal rhythm of melatonin in the gastrointestinal tissues of birds and rodents, with high levels in the dark period. Release of gut melatonin into the general circulation, however, was recorded following tryptophan treatment. Melatonin has direct gastrointestinal functions: it decreased serotonin-induced gut concentration, alleviated serotonin-induced reduction in gastric glandular mucosal blood flow, diminished epithelial sodium absorption, and inhibited the proliferation of jejunal epithelium. The use of a radiolabeled melatonin agonist, 2-[125I]iodomelatonin, has allowed the study of putative melatonin receptors in the gut of duck, mouse, chicken and human. The gastrointestinal 2-[125I]iodomelatonin binding to the duck, chicken and human was rapid, stable, saturable, reversible, specific and with a high affinity. The 2-[125I]iodomelatonin binding sites in the mouse gut were of a much lower affinity. In the duck gut, there was a significant variation in the densities of 2-[125I]iodomelatonin binding sites in different regions of the gut, with the following descending order of density: ileum, jejunum > duodenum, colon > cecum > esophagus. Autoradiographical studies have showed that the highest concentration of 2-[125I]iodomelatonin binding sites was in the villi of the small intestine and also in the mucosal layers of the cecum and colon. In contrast to the diurnal rhythms of 2-[125I]iodomelatonin binding sites reported in other tissues, 2-[125I]iodomelatonin binding in the duck gut showed no daily rhythm. The subcellular distribution of binding sites was in the following descending order: nuclear > microsomal > mitochondrial >> cytosolic fraction. These findings are consistent with a paracrine and/or hormonal action of melatonin in the gastrointestinal tract.