The effect of a 5-HT3 receptor-selective agonist SR57227A was investigated on the outflow of 5-hydroxytryptamine (5-HT) from isolated muscle layer-free mucosal preparations of guinea-pig colon. The mucosal preparations were incubated in vitro and the outflow of 5-HT from these preparations was determined by high-performance liquid chromatography with electrochemical detection. SR57227A (100 μM) produced a tetrodotoxin-resistant and sustained increase in the outflow of 5-HT from the mucosal preparations. The SR57227A-evoked sustained 5-HT outflow was completely inhibited by the 5-HT3 receptor antagonist ramosetron (1 μM). The neuropeptide Y1 receptor antagonist BIBO3304 (100 nM) partially inhibited the SR57227A-evoked sustained 5-HT outflow, but the Y2 receptor antagonist BIIE0246 (1 μM) or the glucagon-like peptide-1 (GLP-1) receptor antagonist exendin-(9−39) (1 μM), showed a minimal effect on the SR57227A-evoked sustained 5-HT outflow. In the presence of BIBO3304 (100 nM) and exendin-(9−39) (1 μM), SR57227A (100 μM) failed to produce a sustained increase in the outflow of 5-HT. The Y1 receptor agonist [Leu31, Pro34]-neuropeptide Y (10 nM), but not GLP-1-(7−36) amide (100 nM), produced a sustained increase in the outflow of 5-HT. We found that 5-HT3 receptor-triggered 5-HT release from guinea-pig colonic mucosa is mediated by the activation of 5-HT3 receptors located at endocrine cells (enterochromaffin cells and peptide YY (PYY)-containing endocrine cells). The activation of both Y1 and GLP-1 receptors appears to be required for the maintenance of 5-HT3 receptor-triggered 5-HT release. It is therefore considered that 5-HT3 receptors located at colonic mucosa play a crucial role in paracrine signaling between enterochromaffin cells and PYY-containing endocrine cells.