In these studies, [ 3 H]epibatidine is used as the radioligand to characterize nicotinic acetylcholine receptors (nAChRs) from bovine adrenal medulla. Specific binding reaches equilibrium within 30 min, and is saturable with a K d value of 0.5 nM. The affinities of several cholinergic agents were determined, including nicotine (K i , 0.2 μM), cytisine (K i , 0.4 μM), carbachol (K i , 4.7 μM), dihydro-β-erythrodine (K i , 33.6 μM), d-tubocurarine (K i , 0.4 μM), 1,1-dimethyl-4-phenyl-piperazinium (K i , 0.8 μM), decamethonium (K i , 234 μM) and methyllycaconitine (K i , 1.3 μM). These values are similar to reported values for recombinant α3β4 nAChRs in transfected cell lines. These studies demonstrate [ 3 H]epibatidine binding to an easily obtainable adrenal membrane preparation and support the characterization of adrenal nAChRs as α3β4* nAChRs.