The occurrence of protein tyrosine nitration under disease conditions is now firmly established and represents a shift from the signal transducing physiological actions of ^•NO to oxidative and potentially pathogenic pathways. Tyrosine nitration is mediated by reactive nitrogen species such as peroxynitrite anion (ONOO^–) and nitrogen dioxide (^•NO₂), formed as secondary products of ^•NO metabolism in the presence of oxidants including superoxide radicals (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document}), hydrogen peroxide (H₂O₂), and transition metal centers. The precise interplay between ^•NO and oxidants and the identification of the proximal intermediate(s) responsible for nitration in vivo have been under controversy. Despite the capacity of peroxynitrite to mediate tyrosine nitration in vitro, its role on nitration in vivo has been questioned, and alternative pathways, including the nitrite/H₂O₂/hemeperoxidase and transition metal-dependent mechanisms, have been proposed. A balanced analysis of existing evidence indicates that (i) different nitration pathways can contribute to tyrosine nitration in vivo, and (ii) most, if not all, nitration pathways involve free radical biochemistry with carbonate radicals (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{CO}}_{3}^{{\bullet}-}\end{equation*}\end{document}) and/or oxo–metal complexes oxidizing tyrosine to tyrosyl radical followed by the diffusion-controlled reaction with ^•NO₂ to yield 3-nitrotyrosine. Although protein tyrosine nitration is a low-yield process in vivo, 3-nitrotyrosine has been revealed as a relevant biomarker of ^•NO-dependent oxidative stress; additionally, site-specific nitration focused on particular protein tyrosines may result in modification of function and promote a biological effect. Tissue distribution and quantitation of protein 3-nitrotyrosine, recognition of the predominant nitration pathways and individual identification of nitrated proteins in disease states open new avenues for the understanding and treatment of human pathologies.