Activated alveolar macrophages secrete both nitric oxide and superoxide in the alveolar lining fluid which combine rapidly to form peroxynitrite, a potent oxidizing agent capable of damaging lipids and proteins in biological membranes. Peroxynitrite (1 mM) plus 100 μM Fe3+EDTA inhibited calf lung surfactant extract (CLSE) from reaching a minimum surface tension below 10 mN/m on dynamic compression. Peroxynitrite and its by-products reacted with the unsaturated lipid components of CLSE, as evidenced by the appearance of conjugated dienes and thiobarbituric acid products, and damaged all surfactant proteins. A mixture of the hydrophobic proteins [surfactant protein B (SP-B) and surfactant protein C (SP-C)] exposed to peroxynitrite became incapable of lowering phospholipid minimum surface tension on dynamic compression. Exposure of SP-A to peroxynitrite decreased its ability to cause lipid aggregation and to act synergistically with SP-B and SP-C in lowering surface tension of surfactant lipids. Western blot analysis of SP-A exposed to peroxynitrite was consistent with fragmentation and polymerization of the 28- to 36-kDa triplet band, and amino acid analysis revealed the presence of significant levels of 3-nitro-L-tyrosine. We conclude that peroxynitrite and its reactive intermediates inhibit pulmonary surfactant function by lipid peroxidation and damaging surfactant proteins.