Diversity of neuronal nicotinic acetylcholine receptor binding was measured using [3H]epibatidine after deletion of alpha7, beta2, or beta4 subunits. [3H]Epibatidine binding is distinctly biphasic. Densities of higher (Kd approximately 0.02 nM) and lower (Kd approximately 5 nM) affinity sites in whole brains of wild-type mice are very similar. Relative sensitivity to inhibition by cytisine or alpha-bungarotoxin was used to evaluate pharmacological subsets of the higher- and lower-affinity sites, respectively. Deletion of each subunit had distinct effects on the binding sites. Deletion of alpha7 did not affect higher-affinity sites but reduced the numbers of lower-affinity sites. This reduction was confined to the [3H]epibatidine binding sites sensitive to inhibition by alpha-bungarotoxin. Deletion of the beta2 subunit had the largest effect. Higher-affinity sites sensitive to inhibition by cytisine were eliminated, and cytisine-resistant sites were reduced. Deletion of the beta2 subunit also significantly reduced the number of lower-affinity sites insensitive to alpha-bungarotoxin. beta4 Gene deletion partially reduced cytisine-resistant and alpha-bungarotoxin-resistant sites with lower and higher affinity for [3H]epibatidine, respectively. Gene deletion in four brain regions (thalamus, hippocampus, superior colliculus, and inferior colliculus) elicited changes generally similar to whole brain. However, relative expression of the binding sites differed among the regions. [3H]Cytisine and 125I-alpha-bungarotoxin binding sites were eliminated by beta2 and alpha7 gene deletion, respectively. These studies establish that the lower-affinity sites represent a structurally diverse set of sites that require expression of either alpha7, beta2, or beta4 subunits and extend and confirm previous classifications of the higher-affinity [3H]epibatidine binding sites.