Inhaled nitric oxide (NO) has been shown to prevent oxidant-induced lung injury in isolated-perfused lung models, whereas NO-derived oxidants may contribute to acute lung injury secondary to hyperoxia. Whether inhaled NO improves or contributes to oxidant-mediated lung injury may depend on the timing of NO administration or how lung injury is assessed. The objective of these studies was to determine whether inhaled NO (20 ppm) was protective or harmful to the different lung barriers when it was administered with 95% O2 for 60 h in Sprague-Dawley rats by measuring fluid transport and permeability to protein across the lung endothelium and the alveolar epithelium. Inhaled NO significantly attenuated the O2-mediated lung endothelial injury and abolished the increase in the bronchoalveolar lavage fluid content of rTI40, a specific and sensitive marker of alveolar epithelial type I cell injury, that occurs secondary to hyperoxia. In conclusion, inhaled NO administered with high concentrations of O2 may protect the lung endothelium and the alveolar epithelium against O2-mediated injury.