Using the fluorescent dye 2′,7′-dichlorodihydrofluorescein (DCF-H2) we investigated the role of glutamate in the production of reactive oxygen species (ROS) in cultured neurons from fetal rat forebrain. The addition of an excitotoxic concentration of glutamate (100 microM) produced a generalized decrease in cellular DCF fluorescence accompanied by local areas of increased fluorescence around the margins of the cell body that could be observed within 2–4 min of glutamate exposure. Increases in fluorescence were dependent on NMDA receptor activation and Ca2+ entry and were blocked by the mitochondrial proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Additional studies suggested that the generalized decrease in fluorescence was due to intracellular acidification. These studies suggest a critical role for mitochondria in the production of ROS in association with glutamate excitotoxicity, and additionally demonstrate the feasibility of measuring the production of ROS at the level of the single cell.