Integration of function between gut regions is mediated by means of hormones and long neuronal reflex pathways. Intestinofugal neurons, which participate in one of these pathways, have cell bodies within the myenteric plexus and project their axons from the gut with the mesenteric nerves. They form excitatory synapses on neurons in prevertebral ganglia that in turn innervate other gut regions. The aim of the present study was to characterise immunohistochemically the synaptic input to intestinofugal neurons. The cell bodies of intestinofugal neurons that project from the distal colon were labelled with Fast Blue that was injected into the inferior mesenteric ganglia. Varicosities surrounding Fast Blue-labelled neurons were analysed for immunoreactivity for the vesicular acetylcholine transporter, vasoactive intestinal peptide, and bombesin. Most intestinofugal neurons were surrounded by nerve terminals immunoreactive for the vesicular acetylcholine transporter; many of these terminals also contained vasoactive intestinal peptide and bombesin immunoreactivity. This combination of markers occurs in axons of descending interneurons. Extrinsic denervation had no effect on the distribution of cholinergic terminals around intestinofugal neurons. A decrease in the number of vesicular acetylcholine transporter and vasoactive intestinal peptide immunoreactive terminals occurred around nerve cells immediately anal, but not oral, to myotomy operations. Consistent with previous physiological studies, it is concluded that intestinofugal neurons receive cholinergic synaptic input from other myenteric neurons, including cholinergic descending interneurons. Thus, intestinofugal neurons are second, or higher, order neurons in reflex pathways, although physiological data indicate that they also respond directly to distension of the gut wall.