5-hydroxytryptamine (5-HT) activates colonic splanchnic afferents, a mechanism by which it has been implicated in generating symptoms in post-infectious and post-inflammatory states in humans. Here we compared mechanisms of colonic afferent activation by 5-HT and mechanical stimuli in normal and inflamed rat colon, and after recovery from inflammation. Colonic inflammation was induced in rats by dextran sulphate sodium. Single fibre recordings of colonic lumbar splanchnic afferents revealed that 58% of endings responded to 5-HT (10-4M) in controls, 88% in acute inflammation (p<0.05) and 75% after 21 days recovery (p<0.05 vs control). Maximal responses to 5-HT were also larger, and the estimated EC50 was reduced from 3.2x10-6M to 8x10-7M in acute inflammation and recovered to 2x10-6M. Responsiveness to mechanical stimulation was unaffected. 5-HT3 receptor antagonism with alosetron reduced responses to 5-HT in controls but not in inflammation. Responses to the mast cell degranulator 48/80 mimicked those to 5-HT in inflamed tissue but not in controls, and more 5-HT containing mast cells were seen close to CGRP containing fibres in inflamed serosa. We conclude that colonic serosal and mesenteric endings exhibit increased sensitivity to 5-HT in inflammation with both an increase in proportion of responders and an increase in sensitivity, which is maintained after healing of inflammation. This is associated with alterations in the roles of 5- HT3 receptors and mast cells.