kappa-Bungarotoxin is a high affinity antagonist of neuronal nicotinic acetylcholine receptors of the alpha 3 subtype. Three sequence segments of the alpha 3 subunit that contribute to forming the binding site for kappa-bungarotoxin were previously located using synthetic peptides corresponding to the complete alpha 3 subunit, i.e., alpha 3(1-18), alpha 3(50-71) and alpha 3(180-201). Here we use single residue substituted peptide analogs of the alpha 3(50-71) sequence, in which amino acids are sequentially replaced by Gly, to determine which residues are important for kappa-bungarotoxin binding activity. Although no single substitution obliterated kappa-bungarotoxin binding, several amino acid substitutions lowered the affinity for kappa-bungarotoxin--i.e., two negatively charged residues (Glu51 and Asp62), and several aliphatic and aromatic residues (Leu54, Leu56, and Tyr63). These results indicate that the interface of the alpha 3 subunit with kappa-bungarotoxin involves primarily hydrophobic interactions, and a few negatively charged residues.