Atractylon, a main sesquiterpenic constituent of Atractylodes rhizomes, was studied for the mechanism of its inhibitory effects on the tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity and lipid peroxidation in primary culture of rat hepatocytes. In the preliminary study, atractylon showed an effective antioxidant property tested by its capacity for quenching 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). Further investigations showed that atractylon at the concentrations of 0.01, 0.1 and 1.0 mg/ml decreased the formation of malondialdehyde (MDA), leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) and repair synthesis of DNA induced by 30-min treatment of t-BHP (1.5 mM) in primary cultured rat hepatocytes. Addition of atractylon also attenuated the genotoxicity of t-BHP evaluated by unscheduled DNA synthesis. The sum of the results suggested that the protective effect of atractylon against oxidative stress induced by t-BHP is via its ability to quench free radicals.