Neuronal bungarotoxin (NBT) is a highly selective, slowly reversible, competitive antagonist of the alpha3beta2 neuronal nicotinic receptor. Contributions to NBT sensitivity are made by both the alpha3 and beta2 subunits. We used a chimeric alpha subunit to demonstrate that the entire alpha3 contribution lies within sequence segment 84-215. Construction and analysis of a series of mutant alpha3 subunits identified seven amino acid residues (Thr143, Tyr184, Lys185, His186, Ile188, Gln198, Ser203) within this region that contribute to NBT sensitivity. Changing Thr143 to lysine, as in alpha2, resulted in a approximately 1000-fold loss of NBT sensitivity. The effect on NBT sensitivity of changing each of the other six residues ranged from 1.8- to 40.5-fold. More extensive mutagenesis demonstrated that Thr143 serves as part of the consensus sequence for glycosylation at N141, and it is this glycosylation that is the determinant of NBT sensitivity. Only serine could substitute for threonine to maintain full NBT sensitivity, and changing Asn141 to alanine resulted in a approximately 300-fold loss of NBT sensitivity. The chimera alpha2-181-alpha3, containing all identified determinants except the glycosylation site, formed receptors insensitive to 300 nM NBT. Installation of threonine to complete the glycosylation consensus site in this chimera conferred NBT sensitivity only 10-fold less than that of wild-type alpha3beta2. These seven determinants of NBT sensitivity are located in close proximity to a series of conserved residues that are common features of all nicotinic receptor binding sites.