1. This study was performed to elucidate the actions of 5-hydroxytryptamine (5-HT) on mesenteric afferent discharge and to determine the receptor-mechanisms responsible for these effects. The activity of mesenteric afferents innervating the mid-jejunum of urethane-anaesthetized rats was recorded with extracellular microelectrodes. The discharge of single nerves within the whole nerve recording was monitored using waveform discriminator software. 2. The intravenous injection of 5-HT produced a complex pattern of afferent activation with two distinct components which could be distinguished both in terms of the response characteristics and the receptors involved. Initially, in 64% of nerve bundles, there was a brief (2.0 +/- 0.1 s) but intense activation of afferent discharge with peak afferent firing increasing with incremental doses of 5-HT. The discharge frequency in seventeen single units from these bundles during the initial response to 10 micrograms 5-HT was 13.0 +/- 1.8 impulses s-1 from a baseline discharge of 1.0 +/- 0.1 impulses s-1. 3. This initial response was mimicked by the 5-HT3 receptor agonist, 2-methyl-5-HT, whereas 5-methoxytryptamine (5-MEOT, 10-100 micrograms) had no comparable effect. Similarly, the initial 4. 5-HT response was completely abolished by the 5-HT3 receptor antagonist, granisetron (0.5 mg kg-1). 5-HT also evoked, in approximately 35% of nerve bundles, a delayed response that single unit analysis showed to be mediated by an entirely different population of afferents from those activated during the initial response. This secondary response to 5-HT was characterized by a more prolonged (> 30 s) but less intense period of afferent activity which was coincident with an increase in intrajejunal pressure, and was mimicked by 5-MEOT (10-100 micrograms). The secondary response to 5-HT and the response to 5-MEOT were significantly attenuated by the 5-HT2A receptor antagonist, ketanserin (0.5 mg kg-1), which had no effect on the initial response. The initial response to 5-HT was unaffected by the L-type calcium channel inhibitor nifedipine (1 mg kg-1) or the N-type calcium channel inhibitor omega-conotoxin GVIA (25 micrograms kg-1). However, the secondary response to 5-HT was significantly reduced after treatment with nifedipine. 5. These results demonstrate that 5-HT activates different populations of afferent fibres innervating the rat jejunum. One population of afferents is activated directly via stimulation of 5-HT3 receptors, while another population responds to 5-HT with a time course consistent with secondary activation of mechanosensitive afferents following 5-HT2A-mediated contractile activity.