We used equilibrium binding analysis to characterize the agonist binding properties of six different rat neuronal nicotinic receptor subunit combinations expressed in Xenopus laevis oocytes. The alpha4beta2 receptor bound [3H]cytisine with a Kdapp of 0.74 +/- 0. 14 nM. The rank order of Kiapp values of additional nicotinic ligands, determined in competition assays, was cytisine < nicotine < acetylcholine < carbachol < curare. These pharmacological properties of alpha4beta2 expressed in oocytes are comparable to published values for the high affinity cytisine binding site in rat brain (alpha4beta2), demonstrating that rat neuronal nicotinic receptors expressed in X. laevis oocytes display appropriate pharmacological properties. Use of [3H]epibatidine allowed detailed characterization of multiple neuronal nicotinic receptor subunit combinations. Kdapp values for [3H]epibatidine binding were 10 pM for alpha2beta2, 87 pM for alpha2beta4, 14 pM for alpha3beta2, 300 pM for alpha3beta4, 30 pM for alpha4beta2, and 85 pM for alpha4beta4. Affinities for six additional agonists (acetylcholine, anabasine, cytisine, 1, 1-dimethyl-4-phenylpiperazinium, lobeline, and nicotine) were determined in competition assays. The beta2-containing receptors had consistently higher affinities for these agonists than did beta4-containing receptors. Particularly striking examples are the affinities displayed by alpha2beta2 and alpha2beta4, which differ in 1,1-dimethyl-4-phenylpiperazinium, nicotine, lobeline, and acetylcholine affinity by 120-, 86-, 85-, and 61-fold, respectively. Although smaller differences in affinity could be ascribed to different alpha subunits, the major factor in determining agonist affinity was the nature of the beta subunit.