Activation of the apoptogenic sphingomyelin-dependent signaling pathway in neuronally differentiated PC12 cells with cell-permeant C2-ceramide resulted in a transient and short-lived emission of reactive oxygen species that was maximal 6 h after the beginning of treatment, followed immediately by nuclear translocation of the transcription factor nuclear factor kappaB. The production of reactive oxygen species was necessary for cell death to occur. The origin of the reactive oxygen species was identified as complex I of the mitochondrial electron transport chain. The mitochondria were not dysfunctional, however. They maintained normal membrane potentials and ATP synthesis until the cells began to die and the cell nuclei to condense and to fragment, approximately 12 h after the beginning of treatment. We conclude that a mitochondrial free radical signal plays a role in the sphingomyelin-dependent transduction pathway. Convergent data from postmortem brain suggest that this signaling pathway may be activated in the dopaminergic neurons that die in patients with Parkinson's disease and would provide a mechanism for oxidative stress implicating the mitochondria, both of which have long been hypothesized to play a role in the pathogenesis of this disease.