Parkinson’s disease is a neurodegenerative disorder characterized by severe motor deficits mainly due to degeneration of dopaminergic neurons in the substantia nigra. Decreased levels of the cell’s most important anti-oxidant, glutathione, have been detected in nigral neurons of Parkinson patients, but it is unknown if they are the cause or merely the consequence of the disease. To elucidate if glutathione depletion causes selective degeneration of nigral dopaminergic neurons, we down-regulated glutathione synthesis in different brain areas of adult rats by a viral vector-based RNAi approach. Decreased glutathione synthesis resulted in progressive degeneration of nigral dopaminergic neurons, while extra-nigral and striatal neurons were significantly less vulnerable. Degeneration of dopaminergic neurons was accompanied by progressive protein aggregate formation and functional motor deficits and was partially rescued by α-synuclein. That the survival of nigral dopaminergic neurons depends on the precise control of glutathione levels was further demonstrated by significant degeneration induced through moderate overproduction of glutathione. Over-expression of either of the two subunits of glutamate–cysteine ligase induced aberrant glutathiolation of cellular proteins and significant degeneration of dopaminergic neurons. Thus, while glutathione depletion was demonstrated to be a selective trigger for dopaminergic neuron degeneration, a glutathione replacement approach as a potential treatment option for Parkinson’s patients must be considered with great care. In conclusion, our data demonstrate that survival of nigral dopaminergic neurons crucially depends on a tight regulation of their glutathione levels and that the depleted glutathione content detected in the brains of Parkinson’s disease patients can be a causative insult for neuronal degeneration.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-010-0791-x) contains supplementary material, which is available to authorized users.