The activity of acetylcholinesterase (AChE) is measured to obtain pathological information about the cholinergic system in various disease states and to assess the effect of AChE inhibitors. Using Ellman's method that is commonly used in such examinations, butyrylcholinesterase inhibitors must be added to measure AChE-specific activity because of low selectivity of AChE toward traditional substrates; however, such inhibitors also inhibit AChE. Therefore, it is desirable to obtain an AChE selective substrate that can be used with the Ellman's method. Here, we synthesized novel AChE substrates, 1-methyl-4-acetylthiomethylpiperidine and 1,1-dimethyl-4-acetylthiomethylpiperidine, and evaluated the hydrolysis rate and AChE selectivity by comparison with the results obtained when traditional substrates were used. The hydrolysis rate of the novel compounds by human AChE was one order of magnitude lower than that of the traditional substrates, acetylthiocholine and acetyl-beta-methylthiocholine, whereas the hydrolysis rate using human butyrylcholinesterase was two orders of magnitude lower than that of the traditional substrates. This indicated that AChE showed selectivity towards the novel substrates which was one order of magnitude higher than that of the traditional substrates. The hydrolysis of the novel compounds in a rat cerebral cortical homogenate and a monkey whole blood was completely inhibited by 1 muM of the specific AChE inhibitor, 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one, indicating the high specificity of AChE towards the novel substrates in a crude tissue sample. From these results, we conclude that the novel compounds developed would be suitable AChE-selective substrates for Ellman's method.