Three radiolabeled nicotinic receptor agonists were examined for their binding characteristics and for inhibition by cholinergic compounds in order to distinguish possible differential affinities for subtypes of neuronal nicotinic acetylcholine receptors. KD and Bmax values for [3H]cytisine, [3H]methylcarbamylcholine and [3H]nicotine were determined from Scatchard analysis using an enriched whole-brain membrane fraction from male Sprague-Dawley rats. Respective KD values were 0.15, 1.07 and 0.89 nM while Bmax values were 99, 64 and 115 fmol/mg protein respectively. All three ligands fit a one-site model of receptor-ligand interaction. Concentration-inhibition curves were used to determine Ki values for 16 cholinergic compounds. The rank order of potencies for displacement of the three ligands was: (-)-cytisine > (-)-nicotine > (-)-lobeline = methylcarbamylcholine > 1,1-dimethyl-4-phenylpiperazinium, (+)-nicotine, dihydro-beta-erythroidine, (+/-)-nornicotine > carbachol > arecoline >> oxotremorine, tetrahydroaminoacridine, AF102B >> (-)-cotinine > RS86 = heptylphysostigmine. Correlations of the affinities of these compounds determined with the three ligands were very near to unity. In contrast, there was a negative correlation of affinities for [3H]cytisine compared to affinities for the muscarinic receptor agonist, [3H]oxotremorine-M, and the muscarinic receptor antagonist, [3H]quinuclidinyl benzoate. Using membranes from whole rat brain yields data suggesting that all three nicotinic ligands bind to the same nicotinic acetylcholine receptor subtype, and are unable to distinguish subtypes of neuronal nicotinic acetylcholine receptor at the level examined.