The motility of the gut depends on the chemicals contained in the lumen, but the stimuli that modify motility and their relationship to enteric neural pathways are unclear. This study examined local inhibitory reflexes activated by various chemical stimulants applied to the mucosa to characterise effective physiological stimuli and the pathways they excite. Segments of jejunum were dissected to allow access to the circular muscle on one half of the preparation whilst leaving the mucosa intact on the circumferentially adjacent half. Chemicals were transiently applied to the mucosa and responses were recorded intracellularly in nearby circular muscle cells. The amino acids L-phenylalanine, L-alanine or L-tryptophan (all 1 mM) evoked inhibitory junction potentials (IJPs) (latency 150-300 ms, amplitude 3 - 8 mV, each N > 6) that were blocked by tetrodotoxin and partially blocked by antagonists of P2X receptors and/or a combination of antagonists at 5-HT₃ and 5-HT₄ receptors. The putative mediators, 5-HT (10 µM), ATP (1 mM) and CCK-8 (1-10 µM), elicited IJPs mediated via 5-HT₃, P2X and CCK B receptors, respectively. Responses were only partially reduced by the effective antagonists. IJPs evoked by electrically stimulating the mucosa were unaffected by antagonists that reduced chemically evoked responses. Both chemically and electrically evoked IJPs were resistant to nicotinic, NK₁, NK₃, AMPA, NMDA, or CGRP receptor blockade. We conclude that mucosal stimulation by amino acids activates local neural pathways whose pharmacology depends on the nature of the stimulus. Transmitters involved at some synapses in these pathways remain to be identified.