A method is presented for the isolation of a 155-kDa protein that possesses phenyl valerate hydrolysis activity in the presence of paraoxon but is inhibited by mipafox; the functional definition of neuropathy target esterase (neurotoxic esterase; NTE). Microsomes, isolated from 18-day-old chicken embryos were treated with phospholipase A2 to solubilize the NTE activity. The extract was then combined with polyoxyethylene W1 detergent and resolved by gel filtration chromatography to yield an active fraction with an approximate mass of 200 kDa. This fraction was further purified by preparative isoelectric focusing and native electrophoresis to yield two separate bands possessing NTE activity. The slower migrating band was highly enriched in a 155-kDa protein that was identified as a source of the NTE activity by affinity chromatography using 3-(9'-mercaptononylthio)-1,1,1-trifluoro-propan-2-one bound to Sepharose CL6B. This represents the first report of the isolation of NTE in its active form and aids in the confirmation of the 155-kDa protein as the most likely candidate for NTE.