Diclofenac is a non-steroidal anti-inflammatory drug that is widely used clinically but side effects associated with the administration of the drug have been reported. The apoptotic effect of the drug has been evaluated in human and rat hepatocytes. Apoptosis was observed after exposure to sub-cytotoxic concentrations of the drug, without overlapping with cell necrosis. Flow cytometric analysis revealed a time- and dose-dependent increase of apoptotic nuclei with sub-diploid DNA content. Caspase 8 and 9 mediate the cell-receptor and the mitochondria-initiated apoptotic pathways, respectively. Inhibition of both caspases prevented activation of downstream caspases, thus indicating that diclofenac at least activates caspase 3 and both effector caspases 8 and 9. The hierarchy of caspase activation by diclofenac was investigated. Analysis of kinetics revealed a simultaneous activation of these caspases that was maximal after 12 hr of exposure to the drug. Inhibitors of MPT, prevented the downstream activation of the caspase cascade, thus showing that diclofenac opened the mitochondrial pore. On the other hand, antioxidants were able to prevent caspase activation by diclofenac, revealing that oxidative stress at the mitochondrial level is in the root of MPT induction and caspase cascade activation. Caspase activation is not mediated by Bid cleavage, suggesting that the cell-receptor pathway seems not to be involved. However, a dose-dependent release of caspase 8 from the mitochondria was observed, indicating that caspase 8 can be processed independently of cell death receptors. Caspases 8 and 9 are very likely the apical caspases in diclofenac-induced apoptosis. In addition, an early dose-dependent increase of bclX(L) expression parallel to the generation of reactive oxygen species in the mitochondria was found. In conclusion, the mitochondrial pathway is very likely the only pathway involved in diclofenac-induced apoptosis, which was related to CYP-mediated metabolism of diclofenac, with the highest apoptotic effect produced by the metabolite 5OH-diclofenac.