Peroxynitrite (ONOO-) is a potent nitrating and oxidizing agent that is formed by a rapid reaction of nitric oxide (NO) with superoxide anion (O2/·-). It appears to be involved in the pathophysiology of many inflammatory and neurodegenerative diseases. It has recently been reported (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) that ONOO- generated at neutral pH from NO and O2/·- (NO/O2/·-) was substantially less efficient than preformed ONOO- at nitrating tyrosine. Here we re-evaluated tyrosine nitration by NO/O2/·- with a shorter incubation period and a more sensitive electrochemical detection system. Appreciable amounts of nitrotyrosine were produced by ONOO- formed in situ (2.9 μM for 5 min; 10 nM/s) by NO/O2/·- flux obtained from propylamine NONOate (CH3N[N(O)NO]- (CH2)3NH2+CH3) and xanthine oxidase using pterin as a substrate in phosphate buffer (pH 7.0) containing 0.1 mM L-tyrosine. The yield of nitrotyrosine by this NO/O2/·- flux was approximately 70% of that produced by the same flux of preformed ONOO- (2.9 μM/5 min). When hypoxanthine was used as a substrate, tyrosine nitration by NO/O2/·- was largely eliminated because of the inhibitory effect of uric acid produced during the oxidation of hypoxanthine. Tyrosine nitration caused by NO/O2/·- was inhibited by the ONOO- scavenger ebselen and was enhanced 2-fold by NaHCO3, as would be expected, because CO2 promotes tyrosine nitration. The profile of nitrotyrosine and dityrosine formation produced by NO/O2/·- flux (2.9 μM/5 min) was consistent with that produced by preformed ONOO-. Tyrosine nitration predominated compared with dityrosine formation caused by a low nanomolar flux of ONOO- at physiological concentrations of free tyrosine (<0.5 mM). In conclusion, our results show that NO generated with O2/·- nitrates tyrosine with a reactivity and efficacy similar to those of chemically synthesized ONOO-, indicating that ONOO- can be a significant source of tyrosine nitration in physiological and pathological events in vivo.