Peroxynitrite (ONOO-) is a powerful oxidant and cytotoxic species formed by the rapid reaction between nitrogen monoxide (nitric oxide, .NO) and superoxide (O2.-). At neutral pH ONOO- is partly protonated and this protonated form, peroxynitrous acid (ONOOH), decomposes rapidly to nitrate, forming (an) intermediate(s) with reactivity similar to .OH and .NO2. Peroxynitrite can hydroxylate and nitrate aromatic rings, and aromatic nitration of phenols such as tyrosine by ONOOH is proposed to proceed via a radical mechanism, with intermediate formation of .NO2. Modification of tyrosine by .NO2 also involves nitration via a radical mechanism. Aromatic nitration of phenols by ONOO- has been shown to be enhanced by superoxide dismutase or Fe(3+)-EDTA, which were proposed to catalyze heterolytic cleavage of ONOOH to form a nitrating species similar to the nitronium ion (NO2+). We investigated possible mechanisms of tyrosine modification by various reactive nitrogen species, including ONOO-, 3-morpholinosydnonimine (SIN-1), and .NO2. Reaction of tyrosine with ONOO- leads to formation of 3-nitrotyrosine and dityrosine, indicating intermediate formation of tyrosyl radicals. The pH dependence of formation of both 3-nitrotyrosine and dityrosine by ONOO- suggests that intermediate formation of ONOOH is required. Qualitatively similar results were obtained when ONOOH was generated continuously by H2O2 and NaNO2 at mildly acidic pH or with SIN-1, a compound which at neutral pH releases both .NO and O2.-, presumably producing ONOO-. However, relatively low yields of nitrotyrosine were obtained with SIN-1, possibly because of competing reactions of tyrosyl radicals with .NO or O2.-. Possible involvement of .NO2 in tyrosine modification by ONOO- was studied using hydroxyl radical scavengers, which can increase the radical yield during decomposition of ONOOH and thereby enhance generation of .NO2. Hydroxyl radical scavengers did not affect tyrosine modification by .NO2 directly and slightly inhibited tyrosine modification by authentic ONOO-. However, when ONOO- was produced at a slower rate, either by SIN-1 or by H2O2/NaNO2 at acidic pH, hydroxyl radical scavengers were found to significantly enhance tyrosine nitration. Our results suggest that ONOO- or ONOO(-)-generating systems induce nitration of tyrosine (or tyrosine residues in proteins) via intermediate formation of tyrosyl radicals and .NO2.