Organophosphorus compounds (OPs) induce neurotoxic disorders through interactions with well-known target esterases, such as acetylcholinesterase and neuropathy target esterase (NTE). However, OPs interact with other esterases of unknown biological function. In soluble chicken brain fractions, three components of enzymatic phenylvalerate esterase activity (PVase) called E α , E β and E γ , have been kinetically discriminated. These components are studied in this work for the relationship with acetylcholine-hydrolyzing activity. When E α PVase activity (resistant PVase activity to 1500μM PMSF for 30min) was tested with different acetylthiocholine concentrations, inhibition was observed. The best-fitting model to the data was the non-competitive inhibition model (Km=0.12, 0.22mM, Ki=6.6, 7.6mM). Resistant acetylthiocholine-hydrolyzing activity to 1500μM PMSF was inhibited by phenylvalerate showing competitive inhibition (Km=0.09, 0.11mM; Ki=1.7, 2.2mM). E β PVase activity (resistant PVase activity to 25μM mipafox for 30min) was not affected by the presence of acetylthiocholine, while resistant acetylthiocholine-hydrolyzing activity to 25μM mipafox showed competitive inhibition in the presence of phenylvalerate (Km=0.05, 0.06mM; Ki=0.44, 0.58mM). The interactions observed between the substrates of AChE and PVase suggest that part of PVase activity might be a protein with acetylthiocholine-hydrolyzing activity.