Neuronal NMDA receptor (NMDAR) activation leads to the formation of superoxide, which normally acts in cell signaling. With extensive NMDAR activation, the resulting superoxide production leads to neuronal death. It is widely held that NMDA-induced superoxide production originates from the mitochondria, but definitive evidence for this is lacking. We evaluated the role of the cytoplasmic enzyme NADPH oxidase in NMDA-induced superoxide production. Neurons in culture and in mouse hippocampus responded to NMDA with a rapid increase in superoxide production, followed by neuronal death. These events were blocked by the NADPH oxidase inhibitor apocynin and in neurons lacking the p47(phox) subunit, which is required for NADPH oxidase assembly. Superoxide production was also blocked by inhibiting the hexose monophosphate shunt, which regenerates the NADPH substrate, and by inhibiting protein kinase C zeta, which activates the NADPH oxidase complex. These findings identify NADPH oxidase as the primary source of NMDA-induced superoxide production.