Patterns of alpha-bungarotoxin (alphaBuTX) binding within the brain of the rat have been studied following one of the two procedures: (1) the intraventricular injection of 125I-labeled toxin followed by a survival period of 1--8 days before aldehyde perfusion, or (2) the incubation of fresh cryostat sections of brain tissue in dilute solutions of radioactive toxin. Appropriate controls with nicotine, curare, atropine and native alphaBuTX established that specific nature of the binding. The principal observations were that toxin binding sites are predominantly associated with central areas of the brain in direct receipt of sensory inputs (the main and accessory olfactory bulbs, superior colliculus, ventral lateral geniculate nucleus, cochlear nuclei, the substantia gelatinosa of the spinal cord and spinal trigeminal nucleus, the principal sensory nucleus of the trigeminal, and the dorsal column nuclei) and with limbic areas of the brain (hippocampus, amygdala, olfactory tubercle, medial mammillary nucleus, and the dorsal tegmental nucleus of Gudden). Toxin was not found to bind to cranial motor nuclei with the exception of the dorsal motor nucleus of the vagus and the nucleus ambiguus. The discrete distribution of clusters of silver grains within the granule layer of cerebellar folia I, IX, and X is described as well as the heavy labeling of the inferior and accessory olivary nuclei. In many areas of the brain silver grains were found to overlie cell bodies. It is suggested that this may reflect the presence of both membrane-bound toxin and internalized ligand following initial binding to a membrane receptor site. An attempt was made to correlate the localization of toxin binding sites with the terminal distributions of previously described cholinergic pathways. There appears to be a reasonably good agreement between the distribution of toxin receptors and proposed sites of cholinergic transmission within the hippocampus, interpeduncular nucleus and cerebellum. In most other cases however, lack of data precluded such correlations. The anatomical relationship of alphaBuTX binding activity and neurotransmitters other than acetylcholine is also considered.