Steamed roots of Rehmannia glutinosa (R. glutinosa) have been traditionally used in Oriental medicine for the treatment of auditory diseases such as tinnitus and hearing loss. To investigate whether the ethanol extract of steamed roots of R. glutinosa (SRG) increases activity of antioxidant enzymes and the level of glutathione (GSH), we measured activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR) and GSH level in HEI-OC1 cells after treatment with 5-50 microg/ml of SRG. The SOD and CAT activities were significantly increased in the presence of SRG compared to the control group. Maximal activities of SOD and CAT were observed in these cells exposed to 10 microg/ml of SRG. The GPX activity also increased dramatically in response to the treatment with SRG in a dose-dependent manner. The GR activity was only increased in the presence of 50 microg/ml of SRG compared to the control group. The level of GSH gradually increased in the presence of 5-50 microg/ml of SRG. In the cytotoxicity test, 5-50 microg/ml of SRG did not show any significant cytotoxicity. These results suggest that the traditional use of R. glutinosa for the treatment of auditory diseases may be explained, in part, by activation of intracellular antioxidant enzyme systems. Further studies are necessary to clarify the active constituents of SRG responsible for such biomolecular activities.