Quantitative autoradiography was used to localize nicotinic [3H]acetylcholine (ACh) binding sites in rat brain. High concentrations of nicotinic [3H]ACh binding sites were observed in the anterior and medial nuclei of the thalamus, the medial habenula and the superficial layer of the superior colliculus. Moderate levels of binding sites were observed in a variety of brain regions such as the frontoparietal cortex and the hippocampus. Low levels of nicotinic ACh sites occurred throughout the hypothalamus and the primary olfactory cortex.