To study the importance of oxygen free radical production by and injury to proximal tubule epithelial cells, an in vitro model was established. Rat renal proximal tubule epithelial cells in primary culture were subjected to normoxic conditions or 60 minutes of hypoxia and 30 minutes of reoxygenation. Under normoxic conditions, these cells produced superoxide radical, hydrogen peroxide, and hydroxyl radical. During hypoxia and reoxygenation, there was an increase in the production of these reactive oxygen species, detected in the extracellular medium, of 252, 226, and 45 percent, respectively. The production rate of superoxide radical was most markedly increased in the first five minutes of reoxygenation. Studies employing 2,7-dichlorofluorescein which fluoresces when oxidized by peroxides revealed a seven-fold increase in cellular fluorescence in cells studied after hypoxia and reoxygenation compared with control cells. That increased production of reactive oxygen species played a role in cellular injury was demonstrated by an increase in lipid peroxidation during hypoxia and reoxygenation, as well as substantial injury during hypoxia and reoxygenation which could be largely prevented by the addition of superoxide dismutase, catalase, dimethylthiourea, or deferoxamine to the cells. These studies demonstrate that proximal tubule epithelial cells produce reactive oxygen species in increased amounts during hypoxia and reoxygenation, and that these reactive oxygen species are injurious to the cells under these conditions.