Tyrosine nitration is becoming increasingly recognized as a prevalent, functionally significant post-translational protein modification that serves as an indicator of nitric oxide (NO)-mediated oxidative inflammatory reactions. Nitration of proteins modulates catalytic activity, cell signaling and cytoskeletal organization. Several reactions mediate protein nitration, and all predominantly depend on NO- and nitrite-dependent formation of nitrogen dioxide, a species capable of nitrating aromatic amino acids, nucleotides and unsaturated fatty acids. Here, we review the mechanisms that mediate in vivo protein nitration and how nitration of specific tyrosine residues impacts on protein function.