Subjecting myogenic H9c2 cells to transient energy deprivation leads to a caspase-independent death with typical features of necrosis. Here we show that the rupture of cytoplasmic membrane, the terminal event in necrosis, is shortly preceded by rapid depolarization of mitochondrial membranes. The rapid deenergization of mitochondria critically depended upon prior generation of reactive oxygen species (ROS) during ATP depletion stage. Accordingly, expression of catalase prevented mitochondrial depolarization and averted subsequent necrosis. Interestingly, trifluoperazine, a compound that protects cells from ischemic insults, prevented necrosis of H9c2 cells through inhibition of ROS production. Other factors that regulated the mitochondrial membrane depolarization and subsequent loss of plasma membrane integrity include a stress kinase JNK activated at early steps of recovery from ATP depletion, as well as an apoptotic inhibitory protein ARC. Accordingly, inhibition of JNK or overexpression of ARC prevented mitochondrial depolarization and rescued H9c2 cells from necrosis. ROS and JNK affected mitochondrial deenergization and necrosis independently of each other since inhibition of ROS production did not prevent activation of JNK, whereas inhibition of JNK did not suppress ROS accumulation. Therefore, JNK activation and ROS production represent two independent pathways that control mitochondrial depolarization and subsequent necrosis of cells subjected to transient energy deprivation. Overexpression of ARC, although preventing mitochondrial depolarization, did not affect either JNK activation or production of ROS. The major heat shock protein Hsp72 inhibited JNK-related steps of necrotic pathway but did not affect ROS accumulation. Interestingly, mitochondrial depolarization and subsequent necrosis can be suppressed by an Hsp72 mutant Hsp72DeltaEEVD, which lacks chaperone function but can efficiently suppress JNK activation. Thus, Hsp72 is directly implicated in a signaling pathway, which leads to necrotic death.