In vitro and in vivo studies evaluated neuropathy target esterase (NTE) inhibition and aging (i.e., loss of reactivation potential) by analytical and technical grade racemic and resolved L-(–) and D-(+) isomers of methamidophos (O,S-dimethyl phosphoramidothioate). For studies in vitro, microsomal protein from phenobarbital-induced livers was isolated from chick embryos and NTE inhibition assays were performed using chick embryo brain homogenate treated with 1 or 5 mM methamidophos (with and without metabolic enzymes); for studies in vivo, hens received 30 to 35 mg/kg methamidophos injected into the pectoral muscle. NTE aging in hens was assessed 24 h later or after 30 min to 1 h incubation in vitro using solutions of potassium fluoride (KF) reactivator. Technical methamidophos produced significantly higher levels of aged-inhibited NTE than analytical methamidophos or isolated optical isomers. In vivo, technical methamidophos produced 61% total NTE inhibition with 18% aged and 43% unaged NTE; hens receiving analytical grade averaged 6% aged, 52% unaged, and 58% total NTE inhibition. Results for 1 mM analytical methamidophos in vitro were 5% aged, 54% unaged, and 59% total inhibition; for 1 mM technical methamidophos, values averaged 11% aged, 50% unaged, and 60% total NTE inhibition. The degree of NTE aging obtained both in vivo and in vitro for the isolated D-(+) and L-(–) isomers never exceeded that obtained using analytical grade. These data indicate that impurities in methamidophos could contribute to OPIDN potential. The in vitro methodology described could be applied to first tier screening for detection of NTE inhibition and aging, thus reducing the need for whole-animal testing for OPIDN.