A free radical gas, nitric oxide NO), has many useful functions when produced under physiological conditions by neurons and endothelial cells. However, excess NO has been reported to exert cytotoxic effects by direct toxicity or by reaction with superoxide. Nitric oxide can react with superoxide to generate peroxynitrite which is as reactive as the hydroxyl radical. This study was attempted to observe formation of peroxynitrite and change in amount of NO synthase(NOS) in reperfused skin flap of rats following ischemia. 3-nitro-L-tyrosine, as direct evidence of peroxynitritemediated tissue damage, as well as endothelial and inducible NO synthase(eNOS and iNOS) were studied in ischemic and reperfused skin using western blot analysis. In addition, HNE-modified proteins, as direct evidence of oxidative tissue damage by reactive oxygen species(ROS), was also evaluated. Skin specimens were obtained over time from island skin flaps(3x3 cm2) of rats under the following two conditions: 1) reperfusion following 5 hours of ischemia, and 2) reperfusion following 10 hours of ischemia. In reperfused skin after 5 hours of ischemia, formation of 3-nitro-L-tyrosine and HNE-modified proteins was decreased 1 hour after reperfusion. However, they were thereafter increased and reached a maximum (3-nitro-L-tyrosine: 142%,HNE-modified proteins:237%) 6 hours after reperfusion. In reperfused skin after 10 hours of ischemia, formation of 3-nitro-L-tyrosine and HNE-modified proteins was increased 3 hours post reperfusion, and reached a maximum (3-nitro-L-tyrosine:178%, HNE-modified proteins: 204%) 6 hours after reperfusion. eNOS and iNOS were decreased 1,3 and 6 hours reperfusion following both 5 and 10 hours of ischemia. These results indicate that peroxynitrite-mediated cytotoxicity is involved in ischemic reperfused skin despite the fact that the amounts of both iNOS and eNOS are decreased.