The gastrointestinal tract contains most of the body's 5-hydroxytryptamine (5-HT) and releases large amounts after meals or exposure to toxins. Increased 5-HT release occurs in patients with irritable bowel syndrome (IBS) and their peak plasma 5-HT levels correlate with pain episodes. 5-HT(3) receptor antagonists reduce symptoms of IBS clinically, but their site of action is unclear and the potential for other therapeutic targets is unexplored. Here we investigated effects of 5-HT on sensory afferents from the colon and the expression of 5-HT(3) receptors on their cell bodies in the dorsal root ganglia (DRG). Distal colon, inferior mesenteric ganglion and the lumbar splanchnic nerve bundle (LSN) were placed in a specialized organ bath. Eighty-six single fibres were recorded from the LSN. Three classes of primary afferents were found: 70 high-threshold serosal afferents, four low-threshold muscular afferents and 12 mucosal afferents. Afferent cell bodies were retrogradely labelled from the distal colon to the lumbar DRG, where they were processed for 5-HT(3) receptor-like immunoreactivity. Fifty-six percent of colonic afferents responded to 5-HT (between 10(-6) and 10(-3) M) and 30 % responded to the selective 5-HT(3) agonist, 2-methyl-5-HT (between 10(-6) and 10(-2) M). Responses to 2-methyl-5-HT were blocked by the 5-HT(3) receptor antagonist alosetron (2 x 10(-7) M), whereas responses to 5-HT were only partly inhibited. Twenty-six percent of L1 DRG cell bodies retrogradely labelled from the colon displayed 5-HT(3) receptor-like immunoreactivity. We conclude that colonic sensory neurones expressing 5-HT(3) receptors also functionally express the receptors at their peripheral endings. Our data reveal actions of 5-HT on colonic afferent endings via both 5-HT(3) and non-5-HT(3) receptors.