1. This study was performed to elucidate the type of afferents that mediate the multiple actions of 5-hydroxytryptamine (5-HT) on mesenteric nerve discharge. Electrophysiological recordings were made from mesenteric afferents innervating the mid-jejunum of the urethane-anaesthetized rat. The discharge of single nerves within the whole nerve recording was monitored using waveform discrimination software. 2. Afferents responded to 5-HT in one of two ways: a short latency, transient excitation mediated by 5-HT3 receptors, or a delayed onset, more prolonged effect that was 5-HT2A receptor mediated. Afferents showing the 5-HT3-mediated response did not respond to luminal distension but were sensitive to intraluminal hydrochloric acid (150 mM) in twenty-eight of twenty-nine experiments. In eight experiments, the 5-HT3-mediated response was reversibly abolished by a 2 min exposure to intraluminal application of local anaesthetic (2 % Xylocaine). 3. Mechanosensitive afferents which responded to distension (< 10 cmH2O) did not show a 5-HT3-mediated response (P = 0.92, n = 14), and maintained this mechanosensitivity after luminal anaesthesia. Mechanosensitive afferents did show a secondary response to 5-HT that was significantly attenuated by atropine (100-200 microg kg-1), whereas hexamethonium (8 mg kg-1) had no effect. 4. In animals whose vagal afferent contribution to their mesenteric nerves had been eliminated by chronic truncal vagotomy, the 5-HT3-mediated response was absent in thirty-six of thirty-six nerve bundles. In contrast, mechanosensitivity to distension and the secondary response to 5-HT could still be evoked. 5. These results suggest that 5-HT stimulates mesenteric afferents by a direct action on 5-HT3 receptors that are present on vagal mucosal afferent terminals. The mucosal afferent response to luminal acid, however, was unaffected by treatment with granisetron (0.5 mg kg-1) indicating that endogenous 5-HT from enterochromaffin cells is not essential for transduction of this luminal signal. In contrast, mechanosensitivity in non-vagal afferents was modulated by 5-HT following an intestinal motor response which was influenced by cholinergic tone.