Existence of a binding site for choline esters with an acyl chain of various sizes was examined by comparing the inhibitory potency of the respective compound. In contrast to acetylcholine, which showed a pure competttive pattern of inhibition, choline esters with an acyl chain of a long size (C≥5) expressed a mixed type of inhibition. Binding of choline esters containing a long chain (C_7-C_(12)) to the hydrophobic region in the active site is deduced from a linear relationship between the K_(iE) value and the size of acyl moiety, and a good hydrophobicity relationship. In addition, the non-competitive component in the inhibition of acetylcholinestemse seems to be due to the interaction of choline esters with both the hydrophobic site and the trimethylammonium-binding site in the active center of the acetylated acetylcholinesterase.