The amount of melatonin present in enterochromaffin cells in the alimentary tract is much higher than that in the central nervous system and melatonin, acting at MT₂ receptors, mediates neural stimulation of mucosal HCO₃^- secretion in duodenum in vivo. We have examined effects of melatonin and receptor ligands on intracellular free calcium ([Ca²⁺]_i) signaling in human and rat duodenal enterocytes. Clusters of interconnecting enterocytes (10-50 cells) were isolated by mild digestion (collagenase/dispase) of human duodenal biopsies or rat duodenal mucosa, loaded with fura-2 acetoxymethyl ester, and attached to the bottom of a temperature-controlled perfusion chamber. Clusters provided viable preparations and respond to stimuli as a syncytium. Melatonin and melatonin receptor agonists 2-iodo-N-butanoyl-5-methoxytryptamine and 2-iodomelatonin (1.0-100 nM) increased enterocyte [Ca²⁺]_i, EC₅₀ of melatonin being 17.0 ± 2.6 nM. The MT₂ receptor antagonists luzindole and DH97 abolished the [Ca²⁺]_i responses. The muscarinic antagonist atropine (1.0 µM) was without effect on basal [Ca²⁺]_i and did not affect the response to melatonin. In the main type of response, [Ca²⁺]_i spiked rapidly and returned to basal values within 4-6 min. In another type, the initial rise in [Ca²⁺]_i was followed by rhythmic oscillations of high amplitude. Melatonin-induced enterocyte [Ca²⁺]_i signaling as well as mucosal cell-to-cell communication may be involved in stimulation of duodenal mucosal HCO₃^- secretion.