Two phenyl valerate hydrolyzing carboxylesterases (EC 3.1.1.1) of hen brain were identified as neurotoxic esterases (NTEA and NTEB) by kinetic analysis of organophosphorus inhibition curves. The activities of both NTE isoenzymes with phenyl valerate (PV) as substrate and their inhibition rate constants were determined in six different animals. In-vivo-application of a single oral dose of 500 mg/kg triorthocresyl phosphate (TOCP) caused 86% inhibition of NTEA and 93% inhibition of NTEB within 24 h. Total NTE activity (NTEA plus NTEB) determined by kinetic analysis shows an excellent correlation (r = 0.989) to NTE activity simultaneously tested with a differential NTE assay. The excellent sensitivity (97%) and high specificity (79%) of the NTE differential test is demonstrated.