[3H]Epibatidine binds to nAChR subtypes in mouse brain with higher (KD approximately 0.02 nM) and lower affinity (KD approximately 7 nM), which can be further subdivided through inhibition by selected agonists and antagonists. These subsets are differentially affected by targeted deletion of alpha7, beta2 or beta4 subunits. Most, but not all, higher and lower affinity binding sites require beta2 (Marks, M.J., Whiteaker, P., Collins, A.C., 2006. Deletion of the alpha7, beta2 or beta4 nicotinic receptor subunit genes identifies highly expressed subtypes with relatively low affinity for [3H]epibatidine. Mol. Pharmacol. 70, 947-959). Effects of functional alpha4 gene deletion are reported here. Deletion of alpha4 virtually eliminated cytisine-sensitive, higher-affinity [3H]epibatidine binding as did beta2 deletion, confirming that these sites are alpha4beta2*-nAChR. Cytisine-resistant, higher-affinity [3H]epibatidine binding sites are diverse and some of these sites require alpha4 expression. Lower affinity [3H]epibatidine binding sites are also heterogeneous and can be subdivided into alpha-bungarotoxin-sensitive and -resistant components. Deleting alpha4 did not affect the alpha-bungarotoxin-sensitive component, but markedly reduced the alpha-bungarotoxin-resistant component. This effect was similar, but not quite identical, to the effect of beta2 deletion. This provides the first evidence that lower-affinity epibatidine binding sites in the brain require expression of alpha4 subunits. The effects of alpha4 gene targeting on receptor function were measured using a 86Rb+ efflux assay. Concentration-effect curves for ACh-stimulated 86Rb+ efflux are biphasic (EC50 values=3.3 microM and 300 microM). Targeting alpha4 produced substantial gene-dose dependent reductions in both phases in whole brain and in most of the 14 brain regions assayed. These effects are very similar to those following deletion of beta2. Thus, alpha4beta2*-nAChRs mediate a significant fraction of both phases of ACh stimulated 86Rb+ efflux.