The present study investigated whether thrombin can induce the production of reactive oxygen species (ROS) through activation of neuronal NADPH oxidase and whether this contributes to oxidative damage and consequently to neurodegeneration. Immunocytochemical and biochemical evidence demonstrated that, in neuron-enriched hippocampal cultures, thrombin induces neurodegeneration in a dose-dependent manner. In parallel, ROS production was evident as assessed by analyzing DCF and hydroethidine. Real-time PCR analysis, at various time points after thrombin treatment, also demonstrated that expression of NADPH oxidase subunits (p47(phox) and p67(phox)) occurs. In addition, Western blot analysis and double-label immunocytochemistry showed an up-regulation in the expression of cytosolic components (Rac 1 and p67(phox)), the translocation of cytosolic proteins (p47(phox) and p67(phox)) to the membrane, and the localization of gp91(phox) or p47(phox) expression in hippocampal neurons of cultures and CA1 layer. The thrombin-induced ROS production, protein oxidation, and loss of cultured hippocampal neurons were partially attenuated by an NADPH oxidase inhibitor and/or by several antioxidants. Collectively, the present study is the first to demonstrate that, in cultured hippocampal neurons, thrombin-induced neurotoxicity is, at least in part, caused by neuronal NADPH oxidase-mediated oxidative stress. This strongly suggests that thrombin can act as an endogenous neurotoxin, and inhibitors of thrombin and/or antioxidants can be useful agents for treating oxidative stress-mediated hippocampal neurodegenerative diseases, such as Alzheimer's disease.