The effect of high concentrations of oxygen on cellular proteins was examined in hepatocytes isolated from rats exposed to 100% oxygen for 0, 24, 48, or 54 h. Previous studies have demonstrated that protein oxidation mediated by mixed-function oxidase systems is accompanied by the formation of protein carbonyl derivatives that can be detected by the formation of protein hydrazone derivatives on treatment with 2,4-dinitrophenylhydrazine (DNPH). In these studies the levels of DNPH-reactive proteins increased steadily during 48 h of continuous oxygen treatment and then decreased to control levels by 54 h. Although the specific activity of two hepatic enzymes, glutamine synthetase and glucose-6-phosphate dehydrogenase, decreased during oxygen treatment, antibody titration of each enzyme indicated that the levels of immunological cross-reactive protein either remained constant or increased slightly during 48 h of oxygen treatment. After 54 h of oxygen exposure the levels of immunologically cross-reactive material were significantly reduced. These results suggest that exposure of rats to high concentrations of oxygen leads to the oxidative inactivation of enzymes and the accumulation of oxidized proteins that are subsequently degraded.