Increased production of reactive oxygen species (ROS) such as superoxide(O2) have been implicated in acute lung injury. The ability to confirm a direct role for superoxide in oxidative injury is limited by its rapid dismutation to other toxic species, e.g. hydrogen peroxide, and the lack of specific markers for O2 activity. Superoxide also is known to react rapidly with nitric oxide (NO) to form peroxynitrite (OONO-). Peroxynitrite nitrates tyrosine residues of proteins to form nitrotyrosine which is a stable product detectable in tissue sections with specific antibodies. We hypothesized that nitrotyrosine staining could be used in lungs with increased superoxide and nitric oxide production to localize the site of superoxide activity. To test this hypothesis, we used a previously established model of lung injury in isolated rabbit lungs produced by ventilating lungs with 95% oxygen and infusing L-arginine (1 mM), the substrate for NO synthase, into the pulmonary artery. When hyperoxic lungs are perfused for 60 minutes following L-arginine, they develop hypertension and edema which is mediated by ROS and NO (Resp. Physiol, vol.100, pp 63-74, 1995). Nitrotyrosine staining was performed in control lungs, hyperoxic lungs, hyperoxic lungs treated with L-arginine, and lungs pretreated with intratracheal Cu,Zn superoxide dismutase(SOD)(25,000 u) followed by hyperoxia and L-arginine. At the end of the experiment, lungs were inflation fixed with paraformaldehyde/gluteraldehyde and paraffin embedded. Lung sections from each treatment group were stained with a monoclonal nitrotyrosine antibody followed by a secondary antibody linked to horseradish peroxidase. Oxygen + L-arginine treated lungs showed strong staining in bronchial and alveolar epithelial cells and alveolar macrophages compared to the low background staining present in the control lungs. Lungs treated with intratracheal SOD did not show nitrotyrosine staining in the epithelial cells or macrophages. These data indicate that the site of ·O2- production where it reacts with NO· to form OONO- can be localized in the lung by the nitrotyrosine antibody. A signal which is inhibitable by SOD is characteristic of·O2-. These data impliate pulmonary epithelial cells as the source of ·O2- in this model of hyperoxic lung injury. This approach may be useful to study mechanisms of acute injury in isolated perfused lungs.