Purified acetylcholinesterases (AChE, EC 3.1.1.7) of organophosphate-susceptible and -resistant populations of lesser grain borer (Rhyzopertha dominica) were characterized. Kinetic studies indicated that the affinity of AChE to the substrates acetylthiocholine, acetyl-(β-methyl)thiocholine, and propionylthiocholine was similar between two populations, but was significantly lower toS-butyrylthiocholine in the resistant population than in the susceptible population. The hydrolyzing efficiency of AChE purified from the resistant population was about 2-fold higher than that from the susceptible population for all substrates investigated. AChE from the resistant population was also 13- and 32-fold less sensitive to inhibition by paraoxon and malaoxon, respectively, than AChE from the susceptible population. However, AChE from the resistant population was about 3-fold more sensitive to carbaryl or chlorpyrifos-methyl oxon and 205-fold more sensitive to carbofuran than the AChE from the susceptible population. These results support our contention that an altered AChE functioning as a target site insensitivity mechanism contributed to malathion resistance in lesser grain borer. Nonetheless, such mechanism does not contribute or rather counteracts chlorpyrifos-methyl resistance in this species.