Ascorbic acid (AA) is a well-known reducing agent; however, under appropriate condition, it can facilitate oxidation. In this study, we investigated the effect of AA on dopamine (DA) and glutathione levels in the nigrostriatal dopaminergic system of male seven-week-old C57BL/6 mice (weight, 23-25 g). Mice were treated with AA (400 mg/kg, i.p.) once per day for four weeks, and changes in tyrosine hydroxylase (TH) immunoreactivity, DA and its metabolites, and glutathione (reduced and oxidized) in the substantia nigra pars compacta (SNpc) and striatum were measured. After repeated AA administration, TH-positive immunoreactivity (TH-IR) decreased compared to the control both in the SNpc and striatum. AA treatment also significantly reduced DA levels, and the reduction of which corresponded to changes in TH expression within the same region. In addition, AA increased DA oxidative metabolism according to increases in the DOPAC/DA ratio both in the SNpc and the striatum, whereas the O-methylation pathway in the striatum remained unchanged. Levels of reduced glutathione (GSH) in both the SNpc and striatum were decreased more in the AA treated group than in the control group. Likewise, levels of total glutathione were also decreased in the corresponding regions. Taken together, our data suggest that repeated AA injection induces dopaminergic neurotoxicity through generation of oxidative stress, and that this toxicity is related to the decline of GSH in both the SNpc and striatum. This neurotoxic mechanism may specifically involve enhancement of the oxidative pathway of DA metabolism through coupling with the antioxidant GSH system of the nigrostriatal dopaminergic system.