Protein nitration occurs as a result of oxidative stress induced by reactive oxygen (ROS) and reactive nitrogen species (RNS). Therefore, protein nitration serves as a hallmark for protein oxidation in vivo. We have previously reported on age dependent protein nitration in cardiac tissue of Fisher 344 BN-F1 rats analyzed by two-dimensional gel electrophoresis; however, only one specific nitration site was identified [Kanski, J., Behring, A., Pelling, J., Schöneich, C., 2005a. Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging. Am. J. Physiol. Heart Circ. Physiol. 288, H371-381]. In the present report, we used solution phase isoelectric focusing (IEF) followed by nanoHPLC-ESI-MS/MS that allowed us to obtain good MS/MS data to identify specific sites of protein nitration in cardiac tissue. As expected, more nitrated proteins were detected in cardiac tissue of old rats, including myosin heavy chain, neurofibromin, tropomyosin and nebulin-related anchoring protein. The post-translational modification of these cytoskeletal proteins may provide some rationale for the age-dependent functional decline of the heart.