Hyperoxia is an important therapy for pulmonary insufficiency, but can cause lung damage, presumably through the actions of reactive oxygen species. In the course of injury, the influx of plasma proteins into the alveolar spaces results in an accumulation of proteinaceous materials in the alveoli. The purpose of the present study was to investigate oxidized proteins present in the alveolar lining fluids of rats exposed to hyperoxia, to identify biomarkers for hyperoxic lung injury. Male Fischer-344 rats were exposed to>95% O2 or room air for 48 h, and proteins in BAL fluids were analyzed. Derivatization with 2,4-dinitrophenylhydrazine (DNPH), separation by gel electrophoresis, and western analysis using an anti-DNP antibody revealed DNPH-reactive protein oxidation products. In both the control and hyperoxic rats, several lavage proteins were DNPH-reactive, but the amounts were greater in the hyperoxic animals. Previously, we found oxidized rat beta-casein in BAL fluids of rats exposed to hyperoxia. Two other DNPH-reactive proteins have now been identified by N-terminal sequencing to be (1) alpha-1-antitrypsin precursor, and (2) glycoprotein pp63, which is a 59 kDa bone sialic acid-containing fetuin precursor. Inactivation of alpha-1-antitrypsin by oxidation of a methionine residue to the sulfoxide is a mechanism for de-inhibition of trypsin, resulting in increased proteolytic activity. However, methionine sulfoxides are not reactive with DNPH, so the oxidation we observe is not identical to the physiological mechanisms for activation of trypsin. Nonetheless, oxidants that are capable of producing DNPH-reactive sites on proteins also oxidize methionine residues [Free Rad Biol Med 22:1235]. Phosphorylated rat hepatic glycoprotein (pp63), which is homologous to human alpha 2-Heremans Scmid-glycoprotein, is an inhibitor of insulin receptor tyrosine kinase activity, essential in insulin signaling. Levels of mRNA for pp63 were not detectable in rat lungs [Matrix 13:331], suggesting that the protein we observed results from filtration of plasma into broncho-alveolar spaces. The significance of the DNPH-reactivity of pp63 is not known at this time, although the reaction could be linked to the oxidation of oligosaccharide side chains or of the protein. Further studies into the specific chemical modifications these proteins incur are needed to determine the specific mechanisms responsible for the oxidations, whether the oxidations result in loss of protein function, and whether accumulation of these oxidized proteins is affected by decreased clearance of these modified proteins.