Acetylcholinesterase (AcChE, EC 3.1.1.7) was isolated from the electric organ of T. nobiliana and treated with the active-site-directed alkylating agent 1-bromo-2-[14C]pinacolone ([14C]BrPin), or with BrPin, which acts initially as a competitive inhibitor, Ki = 0.18 mM, and then inactivates the enzyme, k2 = 1.8 x 10(-4) s-1. AcChE aliquots were digested with trypsin and fractionated by reversed phase high performance liquid chromatography. Inactivation caused a decrease in one absorption peak and an increase in another, identified as the peptide beginning at Ala-222 and extending to Arg-242. 5-Trimethylammonio-2-pentanone, a competitive inhibitor, isosteric with acetylcholine, retarded the inactivation and decreased the quantity of labeled peptide. On sequencing, the 14C label was found associated with Cys-231. This was confirmed by comparison with synthesized S-pinacolonylcysteine, by study of effects of blocking the sequencing by o-phthalaldehyde, and by inactivation by 2,2'-dipyridyl disulfide (2-PDS), a thiol-specific reagent that acts initially as a competitive inhibitor, Ki = 0.042 mM, and then inactivates the enzyme, k2 = 5.0 x 10(-4) s-1. This is retarded by 5-trimethylammonio-2-pentanone, and prior inactivation by 2-PDS prevents subsequent reaction of [14C]BrPin in the active site. BrPin inactivates AcChEs from Electrophorus electricus and from human erythrocyte, but 2-PDS does not. Neither reagent inactivates butyrylcholinesterases from human and horse serum.