Cypermethrin is a synthetic pyrethroid that belongs to a group of insecticides with low mammalian toxicity but high insecticidal activity. The present study was designed to investigate the toxicity of cypermethrin on freshly isolated hepatocytes from male and female rats. Hepatocytes were harvested by a collagenase perfusion technique and were exposed to different concentrations of cypermethrin (100, 200, 400, or 800 ng/2×106 cells) for up to 2 h. Cell viability and the leakage of aspartate transaminase (AST) and alanine transaminase (ALT) were determined throughout the incubation period. The cell viability of the hepatocytes from male and female rats exposed to 400 ng and 800 ng was significantly reduced after 60 and 30 min of incubation, respectively. With cells from female rats, viability was also reduced upon exposure to 200 ng cypermethrin for 2 h. The decrease in cell viability was dose and time dependent. The leakage of ALT and AST was significantly increased with 400 and 800 ng concentrations at 60 and 30 min, respectively. ALT leakage from female hepatocytes was significantly increased at 60 min of incubation with the 200-ng dose, whereas 2 h of incubation was required for the leakage of ALT from the cells of male rats. The present data indicate that cypermethrin has toxic effects on male and female rat hepatocytes in a dose- and time-dependent manner. The data suggest that female rat hepatocytes may be more sensitive to the toxic effects of cypermethrin than male cells.