6-Hydroxydopamine is a neurotoxin commonly used to lesion dopaminergic pathways and generate experimental models for Parkinson disease, however, the cellular mechanism of 6-hydroxydopamine-induced neurodegeneration is not well defined. In this study we have explored how 6-hydroxydopamine neurotoxicity is initiated. We have also investigated downstream signaling pathways activated in response to 6-hydroxydopamine, using a neuronal-like, catecholaminergic cell line (PC12 cells) as an in vitro model system. We have shown that 6-hydroxydopamine neurotoxicity is initiated via extracellular auto-oxidation and the induction of oxidative stress from the oxidative products generated. Neurotoxicity is completely attenuated by preincubation with catalase, suggesting that hydrogen peroxide, at least in part, evokes neuronal cell death in this model. 6-Hydroxydopamine does not initiate toxicity by dopamine transporter-mediated uptake into PC12 cells, because both GBR-12909 and nisoxetine ( inhibitors of dopamine and noradrenaline transporters, respectively) failed to reduce toxicity. 6-Hydroxydopamine has previously been shown to induce both apoptotic and necrotic cell-death mechanisms. In this study oxidative stress initiated by 6-hydroxydopamine caused mitochondrial dysfunction, activation of caspases 3/7, nuclear fragmentation, and apoptosis. We have shown that, in this model, proteolytic activation of the proapoptotic protein kinase C delta (PKC delta) is a key mediator of 6-hydroxydopamine-induced cell death. 6-Hydroxydopamine induces caspase 3-dependent cleavage of full-length PKC delta( 79 kDa) to yield a catalytic fragment ( 41 kDa). Inhibition of PKC delta ( with rottlerin or via RNA interference-mediated gene suppression) ameliorates the neurotoxicity evoked by 6-hydroxydopamine, implicating this kinase in 6-hydroxydopamine-induced neurotoxicity and Parkinsonian neurodegeneration.