Acetylcholinesterases (AChEs) very sensitive to fasciculin inhibition (KiS in picomolar range) have a distinctive group of aromatic amino acids in the peripheral region (Y70, Y121, W279 in Torpedo AChE). Enzymes that lack these amino acids like butyrylcholinesterases (BChEs) or one or two of them like cobra venom, insect and chicken AChEs are 1000 to 1,000,000 times less sensitive. Fasciculin is a non-competitive inhibitor of the hydrolysis of choline and neutral esters by very sensitive AChEs. For the other group of enzymes, differences arise according to the type of substrate. Fasciculin still behaves as a non-competitive inhibitor with choline esters. In contrast, hydrolysis of phenylacetate was unaffected or slightly increased with BChEs and a partial competitive inhibition was observed with cobra venom and chicken enzymes.