5-Hydroxytryptamine (5-HT) is a mediator of chloride ion (Cl - ) secretion in the intestine which can be seen as a rise in short circuit current (I s c ) in the Ussing chamber model. We investigated the 5-HT receptor mediating 5-HT-induced Cl - secretion in the human jejunum in vitro. Jejunal segments obtained from patients having gastric bypass surgery for obesity, were stripped of muscularis and mounted in Ussing chambers and short-circuited. The 5-HT receptor agonist-induced change (Δ) in I s c was recorded in the presence and absence of 5-HT receptor antagonists. The rank order of agonist potency was: 5-HT > 5-methoxytryptamine > renzapride (BRL 24924) > α-methyl-5-HT > > 2-methyl-5-HT. In the presence of Cl - -free media or 100 μM furosemide, 5-HT-induced ΔI s c was significantly reduced. It was also antagonized by ≥ 1 μM tropisetron (a 5-HT 3 /5-HT 4 receptor antagonist) and ≥ 10 nM GR 113808 (a selective 5-HT 4 receptor antagonist) with pA 2 values of 6.5 and 7.9, respectively. Another 5-HT 4 receptor antagonist, SC 53606 (0.1 μM), antagonized the 5-HT-induced response with a pA 2 of 7.3. 5-HT 1 -like/5-HT 2 (methysergide), 5-HT 1 p [N-acetyl-5-hydroxytryptophyl 5-hydroxytryptophan amide (5-HTP-DP)], 5-HT 2 A (ketanserin) and 5-HT 3 (ondansetron) receptor antagonists and tetrodotoxin, had no significant effect on the EC 5 0 for 5-HT. In conclusion, this study demonstrates that in the human muscle-stripped jejunum in vitro, 5-HT induced change in short circuit current is mediated by a 5-HT 4 receptor via a non-neural pathway.