We tested the pathogenic role of O−2 radicals in excitotoxic injury. Inactivation of the TCA cycle enzyme, aconitase, was used as a marker of intracellular O−2 levels, and a porphyrin SOD mimetic was used to scavenge O−2. The selective, reversible, and SOD-sensitive inactivation of aconitase by known O−2 generators was used to validate aconitase activity as a marker of O−2 generation. Treatment of rat cortical cultures with NMDA, KA, or the intracellular O−2 generator PQ2+ produced a selective and reversible inactivation of aconitase, which closely correlated with subsequent cell death produced by these agents. The SOD mimetic, but not its less active congener, attenuated both aconitase inactivation and cell death produced by NMDA, KA, and PQ2+. These results provide direct evidence implicating O2− generation in the pathway to excitotoxic injury.