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Serotonin (5-hydroxytryptamine; 5-HT) containing enterochromaffin (EC) cells may detect chemical or mechanical stimuli in the intestinal lumen and respond with release of 5-HT. The aim of this study was to use real-time electrochemical detection methods to detect release of 5-HT from small numbers of EC cells. In guinea-pig ileum, basal release of 5-HT from the unstimulated, unparalyzed intestine was composed of individual release events (8.4 ± 1.8 events, 0.33 ± 0.06 Hz) of different amplitudes but with similar kinetics. Local compression of the mucosa with the electrode evoked peak 5-HT release of 12.3 ± 2.8 μmol L−1 with a sustained release of 3.0 ± 0.7 μmol L−1. Brief application of acetylcholine (ACh) or carbachol elicited a transient peak (5.7 ± 1.3 μmol L−1 occurring at 35 ± 18 s, n = 9) followed by cyclic release of 5-HT (9.7 ± 2.2 events, 0.40 ± 0.13 Hz, n = 6). This study shows that the release of 5-HT occurs rapidly as individual events from a small number of cells and can reach very high concentrations locally.