Oxidative stress in the brain has been increasingly associated with the development of numerous human neurological diseases. Microglia, activated upon neuronal injury or inflammatory stimulation, are known to release superoxide anion (·O), hydrogen peroxide (H2O2), and nitric oxide (NO), thereby further contributing to oxidative neurotoxicity. The reaction of NO and ·O, forming the toxic peroxynitrite (ONOO−), has been proposed to play a pathogenic role in neuronal injury. However, the interactions between H2O2 and NO during oxidative stress, which may promote or diminish cell death, is less clear. In this study, we explored oxidative neurotoxicity induced by H2O2 plus NO in primary cultures of rat cerebral cortex neurons. As the mechanisms may involve reactions between H2O2 and NO, we monitored the production of ONOO−and reactive oxygen species (ROS) throughout the experiments. Results indicated that the NO donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and H2O2 by themselves elicited neuronal death in a concentration- and time-dependent manner. Sublytic concentrations of H2O2 plus SNAP were sufficient to induce neuronal apoptosis as determined by DNA laddering and fluorescent staining of apoptotic nuclei. Transient ONOO−increase was accompanied by rapid H2O2 decay and NO production, whereas ROS slowly decreased following treatment. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation and the cleavage of caspase-3 were observed. Conversely, inhibition of p38 MAPK and caspase-3 significantly reduced apoptotic death induced by H2O2 plus SNAP. These data suggest that H2O2 and NO act synergistically to induce neuronal death through apoptosis in which activation of p38 MAPK and caspase-3 is involved. © 2003 Wiley-Liss, Inc.