The conditions under which nitric oxide (·NO) may modulate or promote lung injury have not been identified. We hypothesized that ·NO-induced injury results from peroxynitrite, formed by the reaction of ·NO with superoxide. The simultaneous generation of ·NO and superoxide by 3- morpholinosydnonimine (SIN-1, 0.1-2 mM) resulted in oxidation of dihydrorhodamine, a marker of peroxynitrite production, and a dose-dependent decrease in the ability of SP-A to enhance lipid aggregation. Western blot analysis of SIN-1 exposed SP-A samples, overlaid with a polyclonal antibody against nitrotyrosine, were consistent with nitration of SP-A tyrosine residues. Superoxide dismutase (100 U/ml), L-cysteine (5 mM), xanthine oxidase (10 mU/ml) and xanthine (500 μM), or urate (100 μM) prevented the SIN-1-induced dihydrorhodamine oxidation and injury to SP-A. ·NO alone, generated by S-nitroso-N-acetylpenicillamine plus 100 μM L-cysteine, or superoxide and hydrogen peroxide, generated by pterin and xanthine oxidase in the absence of iron, did not damage SP-A or oxidize dihydrorhodamine. We concluded that peroxynitrite, but not ·NO or superoxide and hydrogen peroxide, in concentrations likely to be encountered in vivo, caused nitrotyrosine formation and decreased the ability of SP-A to aggregate lipids.