The present study shows that nitric oxide (NO) irreversibly inhibits purified cytochrome oxidase in a reverse oxygen concentration-dependent manner. The inhibition is dramatically protected by a peroxynitrite scavenger, suggesting that peroxynitrite is formed from the reaction of NO with cytochrome oxidase at low oxygen concentration, and that peroxynitrite is involved in irreversible cytochrome oxidase inactivation. Production of nitroxyl anion or superoxide was tested as potential mechanisms underlying the conversion of NO to peroxynitrite. A nitroxyl anion scavenger potently protected the irreversible inhibition, whereas a superoxide dismutase did not provide protective effect, suggesting that the peroxynitrite was formed from nitroxyl anion rather than the reaction of NO with superoxide.