Alterations in the glutathione system and impairment in energy metabolism have both been implicated in the loss of dopamine neurons in Parkinson's disease. This study examined the importance of cellular glutathione and the involvement of oxidative stress in the loss of mesencephalic dopamine and GABA neurons due to inhibition of energy metabolism with malonate, the reversible, competitive inhibitor of succinate dehydrogenase. Consistent with previous findings, exposure to malonate for 24 h followed by 48 h of recovery caused a dose-dependent loss of the dopamine population with little effect on the GABA population. Toxicity was assessed by simultaneous measurement of the high-affinity uptake of [3H]dopamine and [14C]GABA. Total glutathione content in rat mesencephalic cultures was decreased by 65% with a 24-h pretreatment with 10 μM buthionine sulfoxamine. This reduction in glutathione level greatly potentiated damage to both the dopamine and GABA populations and removed the differential susceptibility between the two populations in response to malonate. These findings point to a role for oxidative stress occurring during energy impairment by malonate. Consistent with this, several spin-trapping agents, α-phenyl-tert-butyl nitrone and two cyclic nitrones, MDL 101,002 and MDL 102,832, completely prevented malonate-induced damage to the dopamine neurons in the absence of buthionine sulfoxamine. The spin-trapping agents also completely prevented toxicity to both the dopamine and GABA populations when cultures were exposed to malonate after pretreatment with buthionine sulfoxamine to reduce glutathione levels. Counts of tyrosine hydroxylase-positive neurons verified enhancement of cell loss by buthionine sulfoxamine plus malonate and protection against cell loss by the spin-trapping agents. NMDA receptors have also been shown to play a role in malonate-induced dopamine cell loss and are associated with the generation of free radicals. Consistent with this, toxicity to the dopamine neurons due to a 1-h exposure to 50 μM glutamate was attenuated by the nitrone spin traps. These findings provide evidence for an oxidative challenge occurring during inhibition of energy metabolism by malonate and show that glutathione is an important neuroprotectant for midbrain neurons during situations when energy metabolism is impaired.