We have used a rat brain-slice preparation to study the effects of some cholinomimetic and amino acid agonists and antagonists on the discharge frequency of neurones in the interpeduncular nucleus (i.p.n.), and on the response of these neurones to electrical stimulation of their main excitatory input, the fasciculus retroflexus of Meynert (f.r.m.). A high proportion of i.p.n. neurones were excited by carbachol, acetylcholine (ACh) and muscarine, but methacholine was less effective. The amino acids L-glutamate and L-aspartate were highly effective stimulants of i.p.n. neurones. The responses to ACh or carbachol were greatly reduced by the nicotinic blocking agents hexamethonium, d-tubocurarine and mecamylamine but only slightly reduced by atropine. The response to muscarine was abolished by low doses of atropine. Alpha-Bungarotoxin did not block the response of i.p.n. neurones to f.r.m. stimulation or to cholinomimetic agonists. The response of i.p.n. neurones to f.r.m. stimulation was not appreciably affected by high doses of nicotinic antagonists or atropine nor was there any enhancement of the response by physostigmine. The amino acid antagonists gamma-D-glutamylglycine (gamma DGG) and 2-amino phosphonovalerate (2-APV) were effective blockers of the response to f.r.m. stimulation and preferentially reduced responses to aspartate while having little effect on responses to glutamate or cholinomimetic agonists. It is concluded that ACh is an unlikely candidate for transmitter in this pathway despite abundant neurochemical evidence in its favour. It is more likely that the transmitter is an excitatory amino acid, probably an aspartate-like substance.