Chloroacetonitrile (CAN) is a disinfection by-product of chlorination of drinking water. Epidemiological studies indicate that it might present a potential hazard to human health. The present work provides an evidence for CAN activation to cyanide (CN-) by myeloperoxidase (MPO)/hydrogen peroxide (H2O2)/chloride (Cl-) system in vitro. Optimum conditions for the oxidation of CAN to CN- were characterized with respect to pH, temperature and time of incubation as well as CAN, MPO, H2O2 and KCl concentrations in incubation mixtures. The kinetic parameters governing the reaction; maximum velocity (Vmax) and Michaelis-Menten constant (Km) were assessed. Oxidation of CAN to CN- by NaOCl alone was shown. Addition of the MPO inhibitors; sodium azide (NaN3), 4-amino benzoic acid hydrazine (ABAH) or indomethacin to the reaction mixtures resulted in a significant decrease in the rate of CAN oxidation. Inclusion of the antioxidant enzyme catalase (CAT) in the incubation mixtures resulted in a significant decrease in the rate of CAN oxidation and CN- formation. Addition of the sulfhydryl compounds; glutathione (GSH), N-acetyl-L-cysteine (NAC), L-cysteine or D-penicillamine significantly enhanced the rate of CN- release. In conclusion, MPO/H2O2/Cl- system has the ability of oxidizing CAN to CN-. The present results represent a novel pathway for CAN activation and might be important in explaining CAN-induced toxicity.