Acetylcholinesterase (AChE) was purified, by affinity chromatography, from adult heads of the housefly (Musca domestica) (GSNU strain) known to have mutated AChE gene. The purified AChE showed a single peak in capillary electrophoresis with an overall yield of 42% representing 957-fold purification and a specific activity of about 290 J.1. moll min/mg. It hydrolyzed acetylthiocholine iodide better than S-butyrylthiocholine iodide or propionylthiocholine iodide. AChE-specific inhibitors, BW284C51 and eserine, significantly inhibited the purified AChE, but a butyrylcholinesterase-specific inhibitor, ethopropazine, did not. The housefly AChE was separated into membrane-bound and soluble forms. In vitro inhibition assays with seven organophophates, except for EPN, showed no significant difference between the crude and the purified AChE. Molecular forms of the housefly AChE did not affect the enzyme sensitivity to chlorpyrifos. Inhibition rate (%) of the AChE from the housefly was shown lower than that of the susceptible housefly strain (SRS) to trichlorfon and chlorpyrifos possibly because of the mutated amino acids of the AChE gene.