The aims of these experiments were to determine in vitro whether colonic distension releases acetylcholine, vasoactive intestinal polypeptide (VIP), and substance P in the guinea pig inferior mesenteric ganglia (IMG) and whether cholinergic and peptidergic mechanosensory nerves projecting from the colon to the IMG receive cholinergic input from other enteric neurons. Colonic distension significantly increased the release of [3H]-acetylcholine, VIP-like immunoreactivity, and substance P-like immunoreactivity in the IMG. Nicotinic receptor blockade in the colon diminished the increase in [3H]acetylcholine release, abolished the increase in VIP-like immunoreactivity release during distension, but had no effect on the release of substance P-like immunoreactivity. Nicotinic receptor blockade in the colon also decreased fast mechanosensory input and significantly reduced by 54% the slow excitatory postsynaptic potential amplitude evoked by colonic distension. The data suggest that enteric cholinergic and VIP mechanosensory neurons that project from the colon to sympathetic neurons in the IMG receive peripheral cholinergic input from other enteric neurons. There was no evidence for enteric cholinergic input to the mechanosensory substance P pathway.