We have localized muscarinic cholinergic receptors in the rat brainstem by a light microscopic autoradiographic method. Initially, we examined the kinetics and specificity of the binding of radiolabeled quinuclidinyl benzilate ([3H]QNB) in slide-mounted, frozen tissue sections to determine that we were labeling the muscarinic cholinergic receptors as they had been described previously in biochemical pharmacological studies. Subsequently, using optimal binding parameters to obtain high specific to nonspecific ratios, we labeled tissue sections and generated autoradiograms by the apposition of emulsion- coated coverslips. We found high concentrations of autoradiographic grains associated with many nuclei in the brainstem including the superior colliculus, nuclei of the lateral lemniscus, pontine nucleus, parabrachial nucleus, trigeminal nerve nucleus (V), the tegmental nuclei, nucleus of the facial nerve (VII), the locus coeruleus, the medial vestibular nucleus, cochlear nucleus, of the tractus solitarius, and nucleus of the hypoglossal nerve (XII). We conclude that muscarinic cholinergic effects of acetylcholine and important in the normal function of both the sensory and motor systems. We speculate on the possible mechanisms of action of certain cholinergic agents, such as the mechanism of anticholinergic compounds when used to treat motion sickness or anticholinesterase poisoning.