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Serotonin (5-HT) containing enterochromaffin (EC) cells of the intestine detect chemical and mechanical stimuli in the lumen and respond by releasing 5-HT on to afferent nerve terminals. Recent electrochemical studies in healthy mucosa have shown that the real-time release of 5-HT is a dynamic and highly regulated process, but how this might change in disease is unknown. Our aim was to characterize real-time uptake and release of 5-HT in a mouse model of colitis and compare it with ELISA measurements of 5-HT. Real-time electrochemical methods were coupled with an ELISA assay to determine the effect on 5-HT availability of a mouse model of inflammation (5% w/v dextran sodium sulphate (DSS) induced colitis). Peak and steady state (SS) 5-HT concentrations (calculated from the oxidation current at +400 mV; amperometry mode) were measured with our without the serotonin reuptake transporter blocker fluoxetine (1 lM) in control and DSS-treated mice. Paired and unpaired data were compared with a one way ANOVA (P < 0.05). In mouse colon, SS release of 5-HT was 1.9 ± 0.6 lM (n = 9) and compression-evoked release was 7.1 ± 2.5 lM (n = 9). In DSS treated mice, the release of 5-HT was significantly increased (SS: 3.4 ± 0.6 lM; peak: 14.7 ± 3.0 lM; n = 11). In control mice, fluoxetine significantly increased peak (9.9 ± lM) but not SS release (2.6 ± 0.4 lM), while in DSS mice both were significantly increased (SS: 7.3 ± 1.2 lM; peak: 23.4 ± 4.1 lM). The effects of fluoxetine in DSS mice were greater than in control. ELISA assays supported these data, showing an increase in 5-HT release detected from inflamed colon (n = 5) compared to control (n = 5) in unstimulated or mechanically stimulated preparations and with or without fluoxetine. The release and uptake of 5-HT from the EC cells of the mouse colon are increased during DSS colitis. Our electrochemical data show that both peak and steady state levels are increased and these changes are mirrored by the ELISA data. In addition, the localised 5-HT concentrations at the site of release, measured using amperometry, are significantly higher than those reported using ELISA techniques. Overall, these data show that during colitis, 5-HT availability will be increased. These raised concentrations may substantially alter the activation or desensitisation of serotonin receptors on afferent nerve terminals.