In these studies, [ 3 H]epibatidine is used as the radioligand to characterize recombinant bovine α3β4 nicotinic acetylcholine receptors (nAChRs) expressed in HEK 293 cells. Specific binding reaches equilibrium quickly and is saturable with a K d value of 0.66 nM. The affinities of the several cholinergic agents were determined, including nicotine (K i , 0.5 μM), cytisine (K i , 0.5 μM), carbachol (K i , 4.1 μM), dihydro-(β)-erythroidine (K i , 43.5 μM), d-tubocurarine (K i , 0.1 μM), 1,1-dimethyl-4-phenylpiperazinium (K i , 0.5 μM), decamethonium (K i , 175 μM) and methyllycaconitine (K i , 0.4 μM). These studies show that the pharmacological characteristics of recombinant bovine α3β4 nAChRs are similar to native bovine α3β4* nAChRs, and indicate that the α5 subunit, if present in the native nAChRs, does not affect ligand affinity.