The binding of [3H]epibatidine, an alkaloid isolated from the skin of an Ecuadorean tree frog, was measured both in brain regions dissected from mouse brain and in tissue sections. Binding to each of 12 brain areas was saturable, but apparently monophasic; no indication of multiple binding sites was obtained. However, inhibition of epibatidine binding by nicotine, acetylcholine, methylcarbachol and cytisine in olfactory bulbs revealed a biphasic pattern consistent with the presence of two sites differentially sensitive to inhibition by these nicotinic agonists. Cytisine displayed the greatest difference in inhibitory potency between the two apparent sites. Subsequent analysis of the inhibition of epibatidine binding by cytisine in membranes prepared from 12 brain areas also suggested the presence of two sites in each brain region. The estimated potency of cytisine at each site was similar in each brain region. However, the proportion of [3H]epibatidine binding sites that were more sensitive to inhibition by cytisine and those sites less sensitive to inhibition by this agonist varied markedly among the brain regions. Quantitative autoradiographic analyses of mouse brain revealed pattern of [3H]epibatidine binding sites less sensitive to inhibition by cytisine that differed markedly from the pattern obtained with [3H]nicotine. Among brain regions demonstrating substantial sites less sensitive to cytisine inhibition were the accessory olfactory nucleus, medial habenula, interpeduncular nucleus, fasciculus retroflexus, superior colliculus, inferior colliculus and the pineal gland. The results indicate that epibatidine binds to at least two distinct nicotinic sites in mouse brain that may represent different nicotinic receptor subtypes, one of which appears to be identical to that measured by the binding of other agonists such as nicotine or cytisine.