Mitochondrial Ca²⁺ uptake and Poly(ADP-Ribose) Polymerase-1 (PARP-1) activation are both required for glutamate-induced excitotoxic neuronal death. Since activation of the glutamate receptors can induce increased levels of reactive oxygen species (ROS), we investigated the relationship of mitochondrial Ca²⁺ uptake and ROS generation, and the possibility that ROS increase is a required signal for PARP-1 activation in cultured striatal neurons. Based on the spatial profile of NMDA-induced ROS generation, we found that only mitochondria showed a significant ROS increase within 30 minutes after NMDA receptor activation. This ROS increase was inhibited by the mitochondrial complex inhibitors rotenone and oligomycin, but not by the cytosolic phospholipase A₂ or xanthine oxidase inhibitors. Mitochondrial ROS generation was also inhibited by both removal of Ca²⁺ from extracellular medium and blockage of mitochondrial Ca²⁺ uptake by either a mitochondrial uncoupler or a Ca²⁺ uniporter inhibitor. Furthermore, both DNA damage and PARP-1 activation induced by NMDA treatment was inhibited by blocking mitochondrial Ca²⁺ uptake or by antioxidants. Our results demonstrate that ROS production during the early stage of acute excitotoxicity derives primarily from mitochondria and is Ca²⁺-dependent. More importantly, the increase of mitochondrial ROS serves as a signal for PARP-1 activation, suggesting that concomitant mitochondrial Ca²⁺ uptake and PARP-1 activation constitute a unified mechanism for excitotoxic neuronal death.