Nitric oxide is a precursor of reactive nitrating species such as peroxynitrite and nitrogen dioxide that modify proteins to generate 3-nitrotyrosine. Many diseases are associated with increased levels of protein-bound nitrotyrosine, and this is used as a marker for oxidative damage. However, the regulation of protein nitration and its role in cell function are unclear. We demonstrate that biological protein nitration can be a specific and dynamic process. Proteins were nitrated in distinct temporal patterns in cells undergoing inflammatory activation, and protein denitration and renitration occurred rapidly in respiring mitochondria. The targets of protein nitration varied over time, which may reflect their sensitivity to nitration, expression pattern, or turnover. The dynamic nature of the nitration process was revealed by denitration and renitration of proteins occurring within minutes in mitochondria that were subject to hypoxiaanoxia and reoxygenation. Our results have implications that are particularly important for ischemia-reperfusion injury.