The enteric serotonin transporter (SERT) plays a critical role in modulating serotonin availability and has been implicated in pathogenesis of various intestinal disorders. To date, SERT expression and function have not been investigated in the human intestine. Current studies were designed to characterize the function, expression, distribution and membrane localization of SERT in the native human intestine. Real time PCR studies showed relatively higher SERT mRNA expression in the human small intestine compared to colon (ileum>>duodenum>>jejunum). Northern blot analysis revealed three mRNA hybridizing species encoding SERT (3.0, 4.9 and 6.8 kb) in the human ileum. Consistent with SERT mRNA expression, SERT immunostaining was mainly detected in the epithelial cells of human duodenal and ileal resected tissues. Notably, SERT expression was localized predominantly to the apical and intracellular compartments and was distributed throughout the crypt villus axis. Immunoblotting studies detected a prominent protein band (~70 kDa) in the ileal apical plasma membranes vesicles (AMVs) isolated from mucosa obtained from organ donor intestine. Functional studies showed that uptake of ³ [H]-serotonin (150 nM) in human ileal AMVs was i) significantly increased in the presence of both Na⁺ and Cl^- ; ii) inhibited (~50%) by the neuronal SERT inhibitor, fluoxetine (10 µM) and by unlabeled 5-HT; iii) exhibited saturation kinetics indicating the presence of a carrier mediated process. Our studies demonstrated differential expression of SERT across various regions of the human intestine and provide evidence for the existence of a functional SERT capable of removing intraluminal serotoinin in human ileal epithelial cells.