Summary:Oxidant stress resulting from excess dopamine (DA) may contribute to the development and progression of Parkinson's disease (PD). Free radicals resulting from the enzymatic metabolism of DA are most often discussed in this regard. However, levodopa (L‐DOPA) and DA can also undergo autooxidation, producing free radicals as well as cytotoxic metabolites. We evaluated the neurotoxic effects of the two stereoisomers of L‐DOPA to differentiate between enzyme‐mediated and autooxidation mechanisms. Various concentrations of D‐ or L‐DOPA (1 mMthrough 10 nM) were added to freshly harvested rostral mesencephalic tegmentum cultures. After 72 h, the cultures were fixed and stained for tyrosine hydroxylase (TH). The number of THimmunoreactive (THir) neurons was then assessed and used as an index of DA neuron survival. Both D‐ and L‐DOPA induced a dose‐dependent loss of THir neurons (F10,21= 135.75, p < 0.0001 andF10,21= 142.53, p < 0.0001, respectively) with ED50values of 10‐5.3and 10‐5.2M, respectively. The doseresponse curves for each drug were not significantly different from one another (F1,43= 0.09, p > 0.05). Moreover, both drugs killed THir as well as non‐THir cells at high concentrations, suggesting a nonspecific toxic effect. These data are most consistent with an enzyme‐independent, autooxidation‐mediated mechanism for DA neuron loss.