In these studies, [(3)H]epibatidine is used as the radioligand to characterize recombinant bovine alpha3beta4 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 microM), cytisine (K(i), 0.5 microM), carbachol (K(i), 4.1 microM), dihydro-(beta)-erythroidine (K(i), 43.5 microM), d-tubocurarine (K(i), 0.1 microM), 1,1-dimethyl-4-phenylpiperazinium (K(i), 0.5 microM), decamethonium (K(i), 175 microM) and methyllycaconitine (K(i), 0.4 microM). These studies show that the pharmacological characteristics of recombinant bovine alpha3beta4 nAChRs are similar to native bovine alpha3beta4* nAChRs, and indicate that the alpha5 subunit, if present in the native nAChRs, does not affect ligand affinity.