In rat brainstem slice preparations, intracellular recording from neurons (n = 39) in the compact formation of the nucleus ambiguus (AMBc) revealed spontaneous and miniature excitatory postsynaptic potentials (EPSPs; n = 11) that, along with acetylcholine-induced depolarization, were enhanced by physostigmine (10 microM; n = 2) and blocked by dihydro-beta-erythroidine 1-5 pmol (n = 4). Retrograde neuronal tracing combined with choline acetyltransferase immunocytochemistry demonstrated that the AMBc receives a projection from a subpopulation of cholinergic neurons in the zona intermedialis reticularis parvicellularis. Electrical stimulation of this region in slices evoked fast EPSPs in AMBc neurons (n = 23) that were inhibited by dihydro-beta-erythroidine 2-5 pmol (n = 8), but not by methscopolamine 1 pmol (n = 2). The present findings strongly support the existence of a cholinergic nicotinic synapse mediating fast transmission in brainstem vagal motoneurons.