This study was undertaken to examine the combined effect of nitric oxide (NO) and hyperoxia on lung edema and Na,K-ATPase expression. Newborn piglets were exposed to room air (FiO2 = 0.21), room air plus 50 ppm NO, hyperoxia (FiO2 ≥ 0.96) or to hyperoxia plus 50 ppm NO for 4–5 days. Animals exposed to NO in room air experienced only a slight decrease in Na,K-ATPase α subunit protein level. Hyperoxia, in the absence of NO, induced both the mRNA and the protein level of Na,K-ATP-ase α subunit and significantly increased wet lung weight, extravascular lung water, and alveolar permeability. NO in hyperoxia decreased the hyperoxic-mediated induction of Na,K-ATPase α subunit mRNA and protein while wet lung weight, extravascular lung water, and alveolar permeability remained elevated. These results suggest that 50 ppm of inhaled NO may not improve hyperoxic-induced lung injury and may interfere with the expression of Na,K-ATPase which constitutes a part of the cellular defense mechanism against oxygen toxicity.