Fibronectin is a large dimeric glycoprotein present in both human plasma and in basement membranes. The latter are specialized extracellular matrices underlying endothelial cells in the artery wall. Peroxynitrous acid (ONOOH) a potent oxidizing and nitrating agent, is formed in vivo from superoxide and nitric oxide radicals by stimulated macrophages and other cells. Considerable evidence supports ONOOH involvement in human atherosclerotic lesion development and rupture, possibly via extracellular matrix damage. Here we demonstrate that Tyr and Trp residues on human plasma fibronectin are highly sensitive to ONOOH with this resulting in the formation of 3-nitrotyrosine, 6-nitrotryptophan and dityrosine as well as protein aggregation and fragmentation. This occurs with equimolar or greater levels of oxidant, and in a dose-dependent manner. Modification of Tyr was quantitatively more significant than Trp (9.1% versus 1.5% conversion with 500μM ONOOH) after accounting for parent amino acid abundance, but only accounts for a small percentage of the total oxidant added. LC-MS studies identified 28 nitration sites (24 Tyr, 4 Trp) with many of these present within domains critical to protein function, including the cell-binding and anastellin domains. Human coronary artery endothelial cells showed decreased adherence and cell-spreading on ONOOH-modified fibronectin compared to control, consistent with cellular dysfunction induced by the modified matrix. Studies on human atherosclerotic lesions have provided evidence for co-localization of 3-nitrotyrosine and fibronectin. ONOOH-mediated fibronectin modification and compromised cell-matrix interactions, may contribute to endothelial cell dysfunction, a weakening of the fibrous cap of atherosclerotic lesions, and an increased propensity to rupture.