Peroxynitrite (ONOO-), the reaction product of superoxide (O2·-) and nitric oxide (·NO), nitrates tyrosine and other phenolics. We report herein that tryptophan is also nitrated by peroxynitrite in the absence of transition metals to one predominant isomer of nitrotryptophan, as determined from spectral characteristics and liquid chromatography-mass spectrometry analysis. At high peroxynitrite to tryptophan ratios, other oxidation products were detected as well. The amount of nitrotryptophan formed from peroxynitrite increased at acidic pH, with an apparent pK(a) of 7.8. High concentrations of Fe3+-EDTA were required to enhance peroxynitrite-induced nitrotryptophan formation, while addition of up to 15 μM Cu/Zn superoxide dismutase had a minimal effect on tryptophan nitration. Cysteine, ascorbate, and methionine decreased nitrotryptophan yield to an extent similar to that predicted by their reaction rates with ground-state peroxynitrite, and typical hydroxyl radical scavengers partially inhibited nitration. Plots of the observed rate constant of nitrotryptophan formation vs tryptophan concentration presented downward curvatures. Thus, the kinetics of metal- independent nitration reactions were interpreted in terms of two parallel mechanisms. In the first one, ground-state peroxynitrous acid nitrated tryptophan with a second-order rate constant of 184 ± 11 M-1 s-1 at 37 °C. The activation enthalpy was 9.1 ± 0.3 kcal mol-1, and the activation entropy was -19 ± 1 cal mol-1 K-1. In the second mechanism, ONOOH*, an activated intermediate derived from trans-peroxynitrous acid formed in a steady state, was the nitrating agent.