Inhaled nitric oxide (NO) is now established as a selective pulmonary vasodilator. Toxicity potential of NO however remains a great concern. The postulated injurious mechanisms include NO-induced activation of matrix metalloproteinases (MMP) with consequent breakdown of the alveolar extracellular matrix. Endothelin-1 (ET-1) is reportedly a marker of acute pulmonary injury. 8-Isoprostanes are lipid peroxidation products of oxyradical-induced cell membrane injury. We hypothesized that lung lavage fluid from animals exposed to hyperoxia and NO will show increased laminin and hydroxyproline contents as early markers of alveolar capillary basement membrane and matrix breakdown. Additionally, we reasoned that lung lavage fluid ET-1 and 8-isoprostane concentration will correlate with laminin and hydroxyproline concentration. To test this hypothesis, we designed the following study. Twenty-eight newborn piglets were randomized into four exposure groups to breathe for five days in a 200-liter exposure chamber. The groups were (i) room air (RA), (ii) RA+50 ppm NO, (RA+NO), (iii) hyperoxia(FiO2≥0.96) (O2), and (iv) hyperoxia + 50 ppm NO(O2+NO). Lung lavage fluid was obtained and analyzed by ELISA to quantitate laminin, ET-1, and 8-isoprostane concentrations. Lavage fluid hydroxyproline was measured by a colorimetric method. Mean hydroxyproline levels were about twenty fold higher in the O2 and O2 + NO group as compared to other groups. ET-1 concentration was higher in the O2 + NO group than in any other group (P <.02). Lung lavage fluid Laminin and 8-Isoprostane concentration did not differ significantly amongst the groups. We conclude, hyperoxia alone or in combination with 50 ppm NO increases lung lavage fluid hydroxyproline levels. This may reflect increased extracellular matrix breakdown. NO did not exacerbate nor ameliorate this effect of oxygen. Elevated ET-1 concentration may reflect inflammatory induction and airway hyperreactivity.