Radioligand binding studies show that cells of the SH-SY5Y human neuroblastoma clonal line express a single class of neuronal/nicotinic alpha-bungarotoxin binding sites (nBgtS). These sites are defined by their ability to bind radioiodinated alpha-bungarotoxin (Bgt) with high affinity (K(D) = 4 nM) and nicotinic ligands with lower (muM) affinities. Radioligand binding studies also show that SH-SY5Y cells express high affinity specific binding sites for (3) H-labeled acetylcholine that could reflect interactions with two classes of sites (K(D') - 1 n/M, K(D'') = 100 nM). These sites are distinguished from nBgtS by their insensitivity to blockade by Bgt and by their ability to bind other nicotinic agonists with high (nM) affinity. (86) Rb(+) efflux studies indicate that SH-SY5Y cells express functional nicotinic acetylcholine receptor (nAChR) ion channels that, like radioagonist binding sites, are insensitive to blockade by Bgt. However, there are far more functional nAChR than high affinity radioagonist binding sites. Functional nAChR have a pharmacological profile expected of ganglia-type receptors composed of nAChR alpha3 and beta4 subunits. Northern blot analysis indicates that genes corresponding to human alpha3, alpha5, beta2, and beta4 subunits are expressed by SH-SY5Y cells. We conclude that SH-SY5Y cells express at least two members of the nAChR family (nBgtS and ganglia-type nAChR) and that high affinity radioagonist binding sites are a subset of functional ganglia-type nAChR.