ABT-418 [(S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole] is a potent and selective agonist at neuronal nicotinic acetylcholine receptors (nAChRs) with cognitive enhancing and anxiolytic activities. [3H]ABT-418 was found to bind with high affinity (KD = 2.85 +/- 0.14 nM) to membranes prepared from rat brain. Binding of [3H]ABT-418 was characterized by rapid association (T1/2 = 1.4 +/- 0.3 min) and dissociation (T1/2 = 2.9 +/- 0.4 min) half-times. The pharmacology of [3H]ABT-418 binding was consistent with an interaction with the putative alpha 4 beta 2 nAChR subtype. The nAChR agonists, (-)-nicotine, (-)-cytisine and (+/-)-epibatidine, displayed a high affinity (Ki = 0.8 +/- 0.1, 0.2 +/- 0.1 and 0.05 +/- 0.01 nM, respectively) for [3H]ABT-418 binding sites, whereas among nAChR antagonists examined, only dihydro-beta-erythroidine competed with high affinity (Ki = 32 +/- 1.5 nM). Although autoradiography studies indicate that the binding distribution of [3H]ABT-418 and (-)-[3H]cytisine are largely identical, there are some brain regions including the striatum, olivary pretectal nucleus and the superior colliculus, in which [3H]ABT-418 demonstrates significantly (P < .05) less binding. The data in the present study demonstrate that [3H]ABT-418 binds with high affinity to a population of binding sites in the rat brain that have the pharmacological characteristics of neuronal nAChRs. [3H]ABT-418 may, therefore, serve as a useful radioligand to further probe the observed differences in pharmacological properties between ABT-418 and other nicotinic agonists in vivo.