The intestinal peristaltic reflex can be elicited by mucosal stimulation or circular muscle stretch. Muscle stretch activates extrinsic, whereas mucosal stimulation activates intrinsic calcitonin gene-related peptide (CGRP)-containing sensory neurons. The present study examined the role of 5-hydroxytryptamine (5-HT) in sensory transmission. A three-compartment preparation of rat colon was used that enables separate measurement of sensory transmitters and modulators. Mucosal stimuli (2-8 brush strokes) caused concurrent increase in 5-HT and CGRP release in proportion to the intensity of stimulation. Release of both 5-HT and CGRP occurred exclusively into the central compartment where the stimuli were applied. Exogenous 5-HT caused a concentration-dependent release of CGRP. Release of CGRP induced by exogenous 5-HT or mucosal stimulation was inhibited by selective 5-HT4 and 5-HT1p antagonists but was not affected by 5-HT1A, 5-HT2, and 5-HT3 antagonists. Ascending contraction and descending relaxation of circular muscle measured in the peripheral orad and caudad compartments, respectively, were also selectively inhibited by 5-HT4 and 5-HT1p antagonists added to the central but not peripheral compartments. In contrast, muscle stretch elicited CGRP but not 5-HT release; the ascending contraction and descending relaxation components of the peristaltic reflex induced by muscle stretch were not affected by 5-HT antagonists. We conclude that 5-HT released by mucosal stimulation initiates the peristaltic reflex by activating 5-HT4/5-HT1p receptors on sensory CGRP-containing neurons.