The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for alpha-bungarotoxin. Using radiolabeled ligand binding techniques, we show that the binding of [H-3]-(+/-)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15 nM and mu M. These classes of sites exist in approximately equal numbers and all [H-3]-(+/-)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses. (c) 2007 Elsevier Inc. All rights reserved.