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Background/Aims: Enterochromaffin (EC) cells are sensors in the intestinal lumen that detect chemical or mechanical stimuli and respond with the release of serotonin (5-HT). 5-HT can activate local reflexes but whether local activation of enteric neural circuits also evoke 5-HT release is unclear. Methods: Recordings were made from full-thickness preparations of guinea pig ileum using electrochemical techniques with carbon fibre electrodes placed in the mucosa to measure local concentrations of 5-HT. The tension in the circular muscle (CM) was recorded with a force transducer. Amplitude and time course of local 5-HT release events and muscle contractions were measured and compared using paired Student’s t-test with Bonferroni’s correction for multiple comparisons. Results: Stretch of the CM caused 5-HT release and reflex contraction of the CM. Focal electrical stimulation of the intestine near to the carbon fibre electrode evoked 5-HT release and caused a local contraction in the ring of CM where the recording site was located. Paralysis of the smooth muscle with papaverine (100 mm; n55), sodium nitroprusside (100 mm; n55) or isoproterenol (1 mm; n55) significantly reduced (o25% of control) the stretch-evoked release of 5-HT. Similarly, isoproterenol (1 mm; n54) or sodium nitroprusside (100 mm; n53), abolished (o10% of control) the electrically evoked release of 5-HT. Atropine (1 mm; n53), which would be expected to block muscarinic input to the EC cell, did not reduce stretch-evoked 5-HT release. Conclusion: The present study provides direct evidence that activated enteric nerves are not responsible for the 5-HT release seen during local reflexes. There was little residual 5-HT release in response to stretch or electrical stimulation of the nerves in paralysed preparations and atropine did not reduce reflex-evoked 5-HT release. Together, these data suggest that mechnical, and not neural, stimuli provide an important excitatory input to the EC cells.