The study on the toxicity of chitosan diethyl phosphate (ChDP), a controlled release insecticide, on the activities of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) in rainbow trout exposed to this pesticide was carried out. It was found that ChDP reduced BuChE activity in O. mykiss by a factor of eight at 6 days, with high fluctuation to the end of the exposition time at 12 days. The in vitro analysis of brain AChE treated with ChDP and Phenamiphos showed that it was competitively inhibited by both organophosphates. The values obtained for Km and Vmax for the AChE-ChDP (Km: 21.23 microM; Vmax: 43.10 micromol/min/g) and AChE-Phenamiphos (Km: 38.62 microM; Vmax: 38.91 micromol/min/g) systems were relatively low compared to values of the AChE (control) system (Km: 62.99 microM; Vmax: 63.29 micromol/min/g). Results reported in this study confirmed that chitosan diethyl phosphate performs similarly to organophosphate pesticides, producing inhibition in cholinesterases in rainbow trout.