Background: Serotonin (5-HT) is a known mediator of intestinal chloride secretion; its importance is elucidated by the several pathologic conditions to which it contributes, including carcinoid syndrome, irritable bowel syndrome, and cholera. Studies have shown that electrogenic chloride secretion can be measured through a change in short-circuit current (Δ)Isc. Through such studies, our lab has previously demonstrated that intestinal chloride secretion occurs at the mucosal level via a non-neural [tetrodotoxin (TTX) - insensitive] 5-HT₃ receptor mediated pathway in rats. The aim of this study was to evaluate the effects of 2-methyl-5-HT, a 5-HT₃ agonist, on TTX-pretreated human intestinal mucosa. Our hypothesis is that selective 2-methyl-5-HT induces intestinal chloride secretion in human small bowel through a non-neural pathway. Methods: Human jejunum, obtained during obesity surgery, was stripped of its seromuscular layer, mounted into Ussing chambers and placed under short-circuit conditions. Tetrodotoxin (TTX) at (1x10^-6 M) alone or TTX plus 2-methyl-5-HT (5x10^-5 M) were added to the chambers. (Δ)Isc was then measured continuously over a 30 minute period with recording of (Δ)Isc maximum value. Results: The data were expressed as (Δ)Isc over time. The 5-HT₃ receptor agonist, 2-methyl-5-HT, significantly increased ΔIsc from 4.8 µA/cm2 to 17.95 µA/cm2 (p<0.01. N=4, repeated measure ANOVA). The stimulatory effect of 2-methyl-5-HT plus TTX was statistically significant when compared to TTX alone. Conclusion: 2-Methyl-5-HT induces chloride secretion by a non-neural, tetrodotoxin-insensitive pathway in human small intestines.