Many pathophysiological processes are associated with oxidative stress and progressive cell death. Oxidative stress is an apoptotic inducer which is known to cause rapid cell death. We show here that a brief oxidative insult (5-min exposure to 400 µM H₂O₂), albeit did not kill H9c2 rat ventricular cells at the time during its presence, triggered an intracellular death cascade leading to delayed time-dependent cell death starting from 1 hr after the insult had been withdrawn and this post-H₂O₂ cell death cumulated gradually reaching a maximum level at 8 hrs after H₂O₂ withdrawal. By comparison, sustained exposure to H₂O₂ caused complete cell death within a narrow time frame (2 hrs). The time-dependent post-H₂O₂ cell death was typical of apoptosis, both morphologically (cell shrinkage and nuclear condensation) and biochemically (DNA fragmentation, extracellular exposure of phosphatidylserines and caspase 3 activation). DCF fluorescent signal showed a time-dependent endogenous increase of ROS production, which was almost abolished by the inhibition of mitochondrial electron transport chain. Application of antioxidants vitamin E or DTT before H₂O₂ addition or after H₂O₂ withdrawal prevented the H₂O₂-triggered progressive ROS production and apoptosis. Sequential appearance of events associated with the activation of mitochondrial death pathway was found, including progressive dissipation of mitochondrial membrane potential, cytochrome c release and late activation of caspase 3. In conclusion, transient oxidative stress triggers an intrinsic program leading to self-sustained apoptosis in H9c2 cells via cumulative production of mitochondrial ROS and subsequent activation of the mitochondrial death pathway. This pattern of apoptosis may contribute to the progressive and long-lasting cell loss in some degenerative diseases.