Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by alveolar inflammation, progressive proliferation of septal cells, increased production of septal matrix, and loss of lung architecture. The process of cellular injury in lung fibrosis is thought to be mediated by oxygen radicals produced by infiltrating inflammatory cells. Peroxynitrite is a potent oxidant produced by the rapid reaction of nitric oxide (NO) and superoxide. We investigated the production of nitrotyrosine, a byproduct of protein nitration by peroxynitrite, and the expression of the enzymes responsible for generating NO, in lungs of patients with IPF and compared them with lungs of normal control subjects. We used immunohistochemistry, histochemistry, and in situ hybridization to study the production of nitrotyrosine and the expression of inducible (iNOS) and constitutive endothelial (eNOS) nitric oxide synthases in 48 lungs of patients with different stages of IPF and 21 normal lungs. In lungs of control subjects, there was little expression of iNOS and nitrotyrosine in the airway epithelium and alveolar macrophages, and abundant expression of eNOS in the airway epithelium and vascular endothelium. By contrast, in lungs of patients with IPF, strong expression of nitrotyrosine and NOS was seen in macrophages, neutrophils, and alveolar epithelium. A significant increase in the expression of these molecules was only seen in lungs of patients with the early to intermediate stage of the disease. The active stage of IPF is associated with increased inflammatory and alveolar expression of nitrotyrosine and NOS. Increased production of NO and peroxynitrite may be responsible for the oxidative damage seen in this disease.