Abstract : Our study of acetylcholinesterase (AcChE) is based on the view that the Beta-trimethylammonio substituent of acetylcholine binds at an uncharged subsite, better termed 'trimethyl' than 'anionic,' and would be more specifically explored by uncharged reagents. AcChE, isolated from Torpedo nobiliana, analyzed fluorimetrically and by Ellman assay, has Km = 0.056 mM, kcat = 4.0 x 103 sec -1 in hydrolysis of acetylthiocholine. 1-Bromo-2-(14C)- pinacolone (14C)-BrPin) inactivates and labels AcChE, and 5-trimethylammonio-2- pentanone (TAP), the isosteric ketone analogue of acetylcholine, prevents this inactivation and reduces 14C introduction. Two 14C are introduced per enzyme unit inactivated, and one 14C is excluded per enzyme unit protected by TAP/ 3- Trimethylammonioacetonph enone, an aromatic analogue of TAP, protects as effectively as TAP. Other benzene and pyridine derivatives, binding at an aryl subsite, inhibit substrate hydrolysis effectively, but protect less effectively against BrPin inactivation, while reducing 14C introduction. Noncompetitive inhibition by tetrasubstituted ammonio inhibitors increase with decreasing substrate reactivity, indicating binding to enzyme-substrate complex (ES), in contrast to the effects of trisubstituted analogues.