Post-translational modification of proteins has been implicated in the loss of physiological functions in aging. The accumulation of modified proteins is species and tissue specific and depends on many processes including elevated oxidant levels, decline in antioxidant protection systems, protein repair and turnover systems. Although elevated levels of protein oxidation and nitration products in aging and age-associated pathology have been reported, the physiological significance of these modifications still needs to be addressed via the characterization of individual protein structure and specific function. We studied the age-dependent modifications of Fisher 344 rat muscle protein glycogen phosphorylase b (PhB) using modern biochemical and proteomic approaches. PhB isolated from muscle of 24 month old rats showed a statistically significant decrease in specific enzyme activity compared to 6 month old animals: 14.7±0.6 vs. 20.6±0.8 U/mg protein, respectively. Reverse-phase HPLC-UV analysis and western blotting of PhB with anti-3-nitrotyrosine antibodies revealed the accumulation of nitrotyrosine in the protein with aging; however, the molar ratio of nitrotyrosine to protein did not exceed 0.16±0.05 for aged animals. HPLC-tandem mass spectrometry of PhB tryptic digests did not show notable selective nitration of protein tyrosine residues. Measuring DTNB-reactive thiols in the protein showed a 10% losses of protein free Cys in aging (ca. 0.8 mol Cys per mol protein). Our data show that F344 rat muscle protein, PhB, undergo oxidative and nitrative modifications under normal biological aging conditions that may account for the loss of protein function, most probably due to irreversible Cys modifications.