Neuronal apoptosis has been implicated as an important mechanism of cell death in acute and chronic neurodegenerative disorders. Ceramide is a product of sphingolipid metabolism which induces neuronal apoptosis in culture, and ceramide levels increase in neurons during various conditions associated with cell death. In this study we investigate the mechanism of ceramide-induced apoptosis in primary cortical neuronal cells. We show that ceramide treatment initiates a cascade of biochemical alterations associated with cell death: earliest signal transduction changes involve Akt dephosphorylation and inactivation followed by dephosphorylation of proapoptotic regulators such as BAD (proapoptotic Bcl-2 family member), Forkhead family transcription factors, glycogen synthase kinase 3-beta, mitochondrial depolarization and permeabilization, release of cytochrome c into the cytosol, and caspase-3 activation. Bongkrekic acid, an agent that inhibits mitochondrial depolarization, significantly reduces ceramide-induced cell death and correlated caspase-3 activation. Together, these data demonstrate the importance of the mitochondrial-dependent intrinsic pathway of caspase activation for ceramide-induced neuronal apoptosis.