Neurofibrillary tangles (NFTs), comprising human intracellular microtubule-associated protein tau, are one of the hallmarks of tauopathies, including Alzheimer's disease. Recently, a report that caspase-cleaved tau is present in NFTs has led to the hypothesis that the mechanisms underlying NFT formation may involve the apoptosis cascade. Here, we show that adenoviral infection of tau into COS-7 cells induces activation of c-jun N-terminal kinase (JNK), followed by excessive phosphorylation of tau and its cleavage by caspase. However, JNK activation alone was insufficient to induce sodium dodecyl sulfate (SDS)-insoluble tau aggregation and additional phosphorylation by GSK-3beta was required. In SH-SY5Y neuroblastoma cells, overexpression of active JNK and GSK-3beta increased caspase-3 activation and cytotoxicity more than overexpression of tau alone. Taken together, these results indicate that, although JNK activation may be a primary inducing factor, further phosphorylation of tau is required for neuronal death and NFT formation in neurodegenerative diseases, including those characterized by tauopathy.