Using whole-cell patch-clamp methods, we examined the hypothesis that serotonin [5-hydroxytryptamine (5-HT)] receptor activation enhances TRPV1 function in mouse colon sensory neurons in lumbosacral dorsal root ganglia, which were identified by retrograde labeling with DiI (1,1'-dioctadecyl-3,3,3',3-tetramethlindocarbocyanine methanesulfonate) injected into multiple sites in the wall of the descending colon. 5-HT increased membrane excitability at a temperature below body temperature in response to thermal ramp stimuli in colon sensory neurons from wild-type mice, but not from TRPV1 knock-out mice. 5-HT significantly enhanced capsaicin-, heat-, and proton-evoked currents with an EC50 value of 2.2 microm. 5-HT (1 microm) significantly increased capsaicin-evoked (100 nm) and proton-evoked (pH 5.5) currents 1.6- and 4.7-fold, respectively, and significantly decreased the threshold temperature for heat current activation from 42 to 38 degrees C. The enhancement of TRPV1 by 5-HT was significantly attenuated by selective 5-HT2 and 5-HT4 receptor antagonists, but not by a 5-HT3 receptor antagonist. In support, 5-HT2 and 5-HT4 receptor agonists mimicked the facilitating effects of 5-HT on TRPV1 function. Downstream signaling required G-protein activation and phosphorylation as intracellularly administered GDP-beta-S [guanosine 5'-O-(2-thiodiphosphate], protein kinase A inhibitors, and an A-kinase anchoring protein inhibitor significantly blocked serotonergic facilitation of TRPV1 function; 5-HT2 receptor-mediated facilitation was also inhibited by a PKC inhibitor. We conclude that the facilitation of TRPV1 by metabotropic 5-HT receptor activation may contribute to hypersensitivity of primary afferent neurons in irritable bowel syndrome patients.