Acetylcholinesterase (AChE, EC 3.1.1.7) purified from the lesser grain borer (Rhyzopertha dominica) was significantly inhibited by higher concentrations of the substrates acetylthiocholine (ATC), acetyl-(β-methyl) thiocholine (AβMTC) and propionylthiocholine (PTC). The efficiency of AChE for hydrolyzing different substrates was ATC > AβMTC > PTC > S-butyrylthiocholine. The enzyme activity was completely inhibited by 10−5 M eserine or BW284C51, but was only partially inhibited by ethopropazine at the same concentration. These results confirmed that the purified enzyme was an typical insect AChE. Non-denaturing and SDS polyacrylamide gel electrophoresis (PAGE) showed only one major molecular form in the purified AChE with a molecular weight of about 107,000 prior to reduction and about 56,000 after reduction, suggesting the homodimer of AChE linked with disulfide bonds.