In order to study where the binding site of cholinergic agents is in the sequence of the alpha-subunit of nicotinic acetylcholine receptor (AChR), we have synthetized 3 peptides with an amino acid sequence corresponding to the following sequences of the alpha-subunit of Torpedo californica AChR: 125-143, 158-167, [Lys] 188-201. For binding studies the peptides were immobilized on Sepharose 4B. Only the peptide [Lys] 188-201 binds 125I-alpha-bungarotoxin (alpha-Bgtx) with Kd of 1.03 microM. The binding of 125I-alpha-Bgtx to the peptide is reduced by 85% after reduction of the S-S bridge present between 192-193 cysteines indicating that an intact disulfide bond is important for toxin binding. The 125I-alpha-Bgtx binding is inhibited by curare, decamethonium, hexamethonium but not by carbamylcholine and Naja naja siamensis alpha-toxin and P15 toxin. All these data provide direct evidence that the sequence 188-201 of the alpha-subunit of AChR binds alpha-Bgtx and that this binding has a pharmacological profile similar to that of nicotinic acetylcholine receptor.