Highly reactive oxygen-containing species may form upon CNS injury and cause oxidative damage to important cellular components, thereby destroying cells. To test this hypothesis, free radical formation following such insults should be characterized first. In this study, we measured the time course of superoxide production following impact injury to the rat spinal cord using a novel microcannula perfusion technique developed by us. Cytochrome c (50 microM in artificial cerebrospinal fluid) was perfused into the rat spinal cord through the cannula inserted laterally into the gray matter of the cord, and reduced cytochrome c was measured from perfusates spectrophotometrically. We found that the levels of superoxide in the extracellular space increased to approximately twice the basal level and remained elevated for over 10 h. Superoxide dismutase (60 U/ml) significantly reduced the elevation of superoxide levels (p = .016) and ferric chloride (0.1 mM)/EDTA (0.25 mM) infused together with cytochrome c completely removed the superoxide measured, validating the measurement of superoxide. The relatively long-lasting formation of superoxide reported herein suggests that removal of superoxide may be a realistic treatment strategy for reducing injury caused by free radicals.