Serotonin is a major component of the inflammatory chemical milieu and contributes to the pain of tissue injury via an action on multiple receptor subtypes. Here we studied mice after genetic or pharmacological disruption of the 5-HT(3) receptor, an excitatory serotonin-gated ion channel. We demonstrate that tissue injury-induced persistent, but not acute, nociception is significantly reduced after functional elimination of this receptor subtype. Specifically, in the setting of tissue injury, the 5-HT(3) receptor mediates activation of nociceptors but does not contribute to injury-associated edema. This result is explained by the localization of 5-HT(3) receptor transcripts to a previously uncharacterized subset of myelinated and unmyelinated afferents, few of which express the proinflammatory neuropeptide substance P. Finally, we provide evidence that central serotonergic circuits modulate nociceptive transmission via a facilitatory action at spinal 5-HT(3) receptors. We conclude that activation of both peripheral and central 5-HT(3) receptors is pronociceptive and that the contribution of peripheral 5-HT(3) receptors involves a novel complement of primary afferent nociceptors.