Respiratory activity of isolated rat brain mitochondria was measured following in vitro exposure to oxygen radicals. The radicals were generated by hypoxanthine and xanthine oxidase in the presence of a suitable iron chelate and caused a severe inhibition of respiration stimulated by phosphate plus ADP (with malate + glutamate as substrate). The damage could be prevented by catalase or high concentrations of mannitol, but not by superoxide dismutase. A similar effect was observed when hypoxanthine and xanthine oxidase were replaced by glucose and glucose oxidase or by hydrogen peroxide. Most of the findings indicate that the hydroxyl radical is the damaging agent. It is concluded that brain mitochondria exposed to oxygen radicals in vitro show an inhibition of respiratory activity similar to that reported by other investigators as occurring in mitochondria in vivo following transient cerebral ischemia. Therefore, oxygen radicals may contribute to this type of cell damage.