Objective To study the oxidative damage to human primary cultured hepatocytes by acute ethanol exposure. Methods Primary hepatocytes were isolated and cultured from human liver tissue. After acute exposure to ethanol of different concentrations and different durations,CYP 2E1 activity in the hepatocytes was measured,meanwhile,glutathione (GSH),malondialdehyde (MDA) in the hepatocytes and aspartate transaminase (AST) in the supernatant were determined to indicate oxidative damage to the cells. Results There was a remarkable dose-and time-dependent relationship between ethanol exposure and CYP 2E1 activity in the human primary cultured hepatocytes. Moreover,there was a time-dependent increase in MDA content in hepatocytes and AST release in supernatants,while the GSH rose at the beginning and later declined significantly. Conclusion There was a markedly oxidative damage to human primary hepatocytes after acute ethanol exposure,and it is clearly related to the activity of CYP 2E1.