Reaction of acetylcholinesterase with methanesulfonyl fluoride is accelerated in the presence of those N-alkyl analogs of acetylcholine that are relatively poor substrates (i.e. slowly hydrolyzed compared to acetylcholine), but is greatly retarded by those that are good substrates, such as acetylcholine itself. The observations are not explained by acetyl enzyme formation. They may be understood if such substrates are first bound at the anionic site and then intermittently contact the esteratic site; attenuation of this second interaction accounts for slow hydrolysis as well as failure to protect the enzyme against methanesulfonyl fluoride, which attacks the esteratic site. Ligands which contain a phenyl ring, including both poor and good substrates, generally protect the enzyme. A phenyl-binding region may therefore be closely associated with the esteratic site. Acceleration of methanesulfonyl fluoride reaction by cationic substrates apparently results from non-specific and normally inhibitory modes of substrate attachment. Like these substrates, reversible cationic inhibitors (which also accelerate the reaction) are now known to be bound in non-specific regions adjacent to the active center. It follows that the activation mechanism is unlikely to involve specific, and normally functional, conformational changes in acetylcholinesterase.