The cytotoxic effect of chlorpyrifos ( CP) on human HepG2 cell lines and the protective role of melatonin were investigated. TD50 of CP for HepG2 cells was also determined. The viability of HepG2 cells decreased with CP treatment in a dose-dependent manner ( P < 0.05). Preincubation with melatonin prior to CP application caused an increase in cell viability ( P < 0.05). TD50 of CP for HepG2 was determined as 84.5 mu g/mL. A 1-hour melatonin treatment caused a decrease in TD50 from 84.5 to 34.1 mg/mL. The level of thiobarbituric acid reactive substance ( TBARS) and the activities of superoxide dismutase ( SOD), glutathione peroxidase ( GSH-Px) and catalase ( CAT) were determined in cell lines with or without melatonin administration to find out the possible mechanism of melatonin. CP caused a significant decrease in SOD, GSH-Px and CAT activities and an increase in TBARS level ( P < 0.05). Pre-incubation of cells with melatonin prevented an increase in TBARS. Melatonin also reduced the CP-caused inhibition of the activities of GSH-Px and CAT ( P < 0.05). It was suggested that CP shows a cytotoxic effect on HepG2 cell lines and melatonin can suppress cytotoxicity caused by CP with its antioxidant properties. Melatonin also reduces TD50 of CP for HepG2 cell lines.