Since melatonin and putative melatonin receptors can be detected in a variety of peripheral tissues, direct endocrine and paracrine actions of melatonin on the physiological functions of different organ systems in response to internal and external stimuli probably exist. As an extension of our earlier work on the pharmacological characterization of 2-[125I]iodomelatonin binding sites in the duck jejunum, the regional and diurnal variations of melatonin and putative melatonin receptors of different segments of the duck gastro-intestinal tract were studied in an attempt to understand the role of melatonin in the physiology of the digestive system. Although no significant effects of diurnal variation and pinealectomy on the regional distribution of melatonin were observed, significant regional variations of melatonin levels were detected with decreasing levels as follows: colon > oesophagus, caecum > duodenum, jejunum > ileum. The densities of melatonin binding sites showed a significant variation between different intestinal regions at either mid-light or mid-dark, with the following descending order: ileum, jejunum > duodenum, colon > caecum > oesophagus. Analysis of the distribution of melatonin binding sites in the wall of the intestine revealed maximal binding in the mucosa and minimal binding in the muscular layers of the jejunum. Similar results were obtained for other intestinal regions as revealed by autoradiography. No significant changes in the affinities of melatonin binding sites were detected between different regions and tissue layers of the alimentary canal. Moreover, the densities and affinities of melatonin binding sites among different regions of the gut exhibited no significant diurnal variations. The demonstration of regional variations in melatonin levels and the density of melatonin binding sites along the gastro-intestinal tract, with a concentration of the putative melatonin receptors in the mucosal layer, suggests a possible direct action of melatonin in the regulation of fluid/electrolyte transport and nutrient absorption in the gut.