We stably transfected human embryonic kidney cells (HEK 293 cells) with genes encoding rat neuronal nicotinic receptor alpha2, alpha3, or alpha4 subunits in combination with the beta2 or beta4 subunit to generate six cell lines that express defined subunit combinations that represent potential subtypes of rat neuronal nicotinic acetylcholine receptors (nAChRs). These cell lines were designated KXalpha2beta2, KXalpha2beta4, KXalpha3beta2, KXalpha3beta4, KXalpha4beta2, and KXalpha4beta4. The Kd values of [3H](+/-)epibatidine ([3H]EB) binding to membranes from these six cell lines ranged from approximately 0.02 to 0.3 nM. The pharmacological profiles of the agonist binding sites of these putative nAChR subtypes were examined in competition studies in which unlabeled nicotinic ligands, including 10 agonists and two antagonists, competed against [3H]EB. Most nicotinic ligands examined had higher affinity for the receptor subtypes containing the beta2 subunit compared with those containing the beta4 subunit. An excellent correlation (r > 0.99) of the binding affinities of the 10 agonists was observed between receptors from KXalpha4beta2 cells and from rat forebrain tissue, in which [3H]EB binding represents predominantly alpha4beta2 nAChRs. More important, the affinities (Ki values) for the two tissues were nearly identical. The densities of the binding sites of all six cell lines were increased after a 5-day exposure to (-)-nicotine or the quaternary amine agonist carbachol. These data indicate that these cell lines expressing nAChR subunit combinations should be useful models for investigating pharmacological properties and regulation of the binding sites of potential nAChR subtypes, as well as for studying the properties of nicotinic compounds.