Gastrointestinal serotonin (5-HT) and melatonin are two closely related neuromodulators which are synthesised in the enterochromaffin cells of the intestinal epithelium and which have been shown to be involved in the physiopathology of the gastrointestinal tract. The effects of 5-HT depend on 5-HT availability which is, in part, modulated by the serotonin transporter (SERT). This transporter provides an efficient 5-HT uptake after release and is expressed in the membrane of the enterocytes. Although the origin and effects of 5-HT and melatonin are similar, the interrelationship between them in the gastrointestinal tract is unknown. The main aim of this study was to determine whether melatonin affects SERT activity and expression, and, if so, to elucidate the mechanisms involved. Caco-2 cell line was used to carry out the study as these cells have been shown to endogenously express SERT. The results showed that melatonin inhibits SERT activity by affecting both V(max) and kt kinetic constants although SERT synthesis or intracellular trafficking did not appear to be affected. The melatonin effect seemed to be independent of melatonin receptors MT(1) and MT(2) and protein kinase C and cAMP intracellular pathways. Our results suggest that the inhibition of SERT might be due to a catalytic effect of melatonin on the allosteric citalopram-sensitive site in SERT. This study shows, for the first time, that melatonin modulates SERT activity, thus demonstrating the feedback system between melatonin and the serotoninergic system in the gastrointestinal tract.