Background: Enterochromaffin (EC) cell-derived 5-hydroxytryptamine (5-HT) is a mediator of toxin-induced reflexes, initiating emesis via vagal and central 5-HT₃ receptors. The amine is also involved in gastrointestinal (GI) reflexes that are prosecretory and promotile, and recently 5-HT's roles in chemosensation in the distal bowel have been described. We set out to establish the efficacy of 5-HT signaling, local 5-HT levels and pharmacology in discrete regions of the mouse small and large intestine. We also investigated the inter-relationships between incretin hormones, glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) and endogenous 5-HT in mucosal and motility assays. Methods: Adult mouse GI mucosae were mounted in Ussing chambers and area-specific studies were performed to establish the 5-HT₃ and 5-HT₄ pharmacology, the sidedness of responses, and the inter-relationships between incretins and endogenous 5-HT. Natural fecal pellet transit in vitro and full-length GI transit in vivo were also measured. Key Results: We observed the greatest level of tonic and exogenous 5-HT-induced ion transport and highest levels of 5-HT in ascending colon mucosa. Here both 5-HT₃ and 5-HT₄ receptors were involved but elsewhere in the GI tract epithelial basolateral 5-HT₄ receptors mediate 5-HT's prosecretory effect. Exendin-4 and GIP induced 5-HT release in the ascending colon, while L cell-derived PYY also contributed to GIP mucosal effects in the descending colon. Both peptides slowed colonic transit. Conclusions & Inferences: We provide functional evidence for paracrine interplay between 5-HT, GLP-1 and GIP, particularly in the colonic mucosal region. Basolateral epithelial 5-HT₄ receptors mediated both 5-HT and incretin mucosal responses in healthy colon.