The inhibition kinetics and stereospecificity of the chiral nerve agent derivative O,S-diethylphenylphosphonothioate (DEPP) were examined for two forms of acetylcholinesterase (human and eel) and equine butyrylcholinesterase. Both S- and R-DEPP are poor inhibitors of eel AChE (IC50 150μM), consistent with a large, nondiscriminatory binding interaction in the active site of this enzyme. However, S-DEPP is active against human and equine AChE with low μM IC50s. DEPP stereospecificities (S/R) toward these enzymes are moderate (20) relative to other cholinesterase-organophosphate (OP) systems. Pralidoxime, a common rescue agent, affects a modest recovery of both hAChE and eqBChE from treatment with S-DEPP. This result is consistent with expected chemical modification by DEPP and indicates that a measurable amount of the enzyme-phosphonate adduct does not undergo aging. Kinetic analysis of inhibition of both hAChE and eqBChE by S-DEPP yields KI values near 8μM and k2 values of about 0.10min-1. In both cases, the reaction is practically irreversible. Second order rate constants calculated from these values are similar to those obtained previously using other thio-substituted OPs with bulky groups. Since BChE has a more accommodating acyl pocket than AChE, the similar behaviors of both enzymes toward S-DEPP is notable and is likely a reflection of the weakened potency of DEPP relative to chemical warfare agents.