Isolated segments of guinea-pig small intestine were set up in a partitioned bath to study the enteric excitatory reflex evoked by distension. The gut was distended by a rubber balloon inserted at the aboral end and contractions of the circular muscle were recorded at the oral end. The oral and aboral ends of the gut were separated by an intermediate compartment of the bath. Inflation of the intraluminal balloon with 0.075-0.35 ml water elicited reproducible and distension-dependent contraction. This enteric orally directed (ascending) excitatory reflex was abolished by tetrodotoxin irrespective of the compartment in which it was applied. Hyoscine (0.3 μM) almost abolished the enteric excitatory reflex when it was applied to the oral compartment. This indicates that the transmission from the final motor neurons to the circular muscle is mainly cholinergic, acting via muscarinic receptors. Hyoscine had no effect on the enteric excitatory reflex when added to the intermediate compartment. When hyoscine was added to the aboral compartment, it decreased the enteric excitatory reflex elicited by low distension stimuli to 70% of control and decreased the enteric excitatory reflex elicited by higher distension stimuli to 95% of control. This indicates that ganglionic transmission involving muscarinic receptors at the site of distension in the aboral bath contributes to the enteric excitatory reflex. Hexamethonium (100 μM) greatly depressed the enteric excitatory reflex when applied to any compartment indicating that nicotinic transmission is most important in the afferent, intermediate and efferent components of the reflex and that the reflex pathway involves a polysynaptic chain of cholinergic interneurons. With both cholinergic receptor antagonists in the aboral compartment, the enteric excitatory reflex elicited by 0.3 ml distension was still 38% of the control response indicating that a significant component of the reflex in the vicinity of the initiation of the reflex is mediated by non-cholinergic ganglionic transmission. It is proposed that the enteric excitatory reflex involves mechanosensory neurons, which, depending on the intensity of the stimuli, release acetylcholine and a non-cholinergic transmitter to provide fast nicotinic, slow muscarinic, and slow non-cholinergic inputs to cholinergic interneurons. These form a polysynaptic, orally directed, pathway which utilize fast nicotinic transmission and activates the final cholinergic motor neurons which contract the circular muscle via muscarinic receptors.