Ascorbic acid (AA) is a well-known antioxidant. It also has pro-oxidant effects, however, in the presence of free transition metals. Because of the pro-oxidant effects of AA, dehydroascorbic acid (DHA), an oxidized form of AA, has been used as a substitute for AA. DHA has been shown recently to have a protective effect in an experimental stroke model. This study was carried out to determine if DHA has different effects from AA on hydrogen peroxide (H2O2)-induced oxidative cell death in primary astrocytes. DHA was found to prevent cell death and reverse mitochondrial dysfunction after exposure to H2O2. DHA significantly increased the glutathione peroxidase (GPx) and glutathione reductase (GR) activities 1 hr after H2O2 exposure. Moreover, DHA not only reversed the decrease in the glutathione (GSH) levels, but also significantly enhanced it by stimulating the pentose phosphate pathway (PPP) 15 hr after H2O2 exposure. DHA also reduced production of reactive oxygen species (ROS) after H2O2 exposure. In contrast, AA accelerated H2O2-induced cell death. To determine if the pro-oxidant effect of AA is related to iron, the effect of AA on cell death was examined using an iron chelator, desferrioxamine. Even though co-pretreatment with AA and desferrioxamine could abrogate the aggravating effects of AA on H2O2-induced cell death at early stages, it could not prevent H2O2-induced cell death over a 24-hr period. These results suggest that DHA has distinct effects from AA and prevent H2O2-induced cell death by increasing the GSH levels mediated by the GPx and GR activities and PPP. © 2005 Wiley-Liss, Inc.