To characterize the structure of the agonist-binding site of the Torpedo nicotinic acetylcholine receptor (AChR), we have used [3H]acetylcholine mustard [( 3H]AChM), a reactive analog of acetylcholine, to identify residues contributing to the cation-binding subsite. Reaction of [3H]AChM, in its aziridinium form, with AChR-rich membrane suspensions, resulted initially in reversible, high affinity binding (K approximately 0.3 microM) followed by slow alkylation of the acetylcholine-binding site. Incorporation of label into AChR alpha-subunit was inhibited by agonists and competitive antagonists, but not by noncompetitive antagonists, and reaction with 3 microM [3H]AChM for 2 h resulted in specific alkylation of 0.6% of alpha-subunits. Within the alpha-subunit, greater than 90% of specific incorporation was contained within an 18-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Val-46 and containing N-linked carbohydrate. To identify sites of specific alkylation, [3H]AChM-labeled alpha-subunit was digested with trypsin, and the digests were fractionated by reverse phase high pressure liquid chromatography. Specifically labeled material was recovered within a single peak containing a peptide extending from Leu-80 to Lys-107. NH2-terminal amino acid sequencing revealed specific release of 3H in cycle 14 corresponding to alpha-subunit Tyr-93. Identification of Tyr-93 as the site of alkylation was confirmed by radiosequence analysis utilizing o-phthalaldehyde to establish that the released 3H originated from a peptide containing prolines at residues 2 and 9. Because [3H]AChM contains as its reactive group a positively charged quaternary aziridinium, alpha-subunit Tyr-93 is identified as contributing to the cation-binding domain of the AChR agonist-binding site. The selective reaction of [3H]AChM with tyrosyl rather than acidic side chains indicates the importance of aromatic interactions for the binding of the quaternary ammonium group, and the lack of reaction with the tyrosyl or acidic side chains within alpha 190-200 emphasizes the selective orientation of acetylcholine within its binding site.