During the glycation reaction between reducing sugars and free amino groups of proteins, alpha-dicarbonyl compounds, such as glyoxal, methylglyoxal, and deoxyglucosones, are produced. These compounds are more reactive than the parent sugars for reacting with amino groups of proteins to form inter-and intra-molecular cross-links of proteins and stable advanced end products that are known to accumulate with aging, diabetes mellitus, Alzheimer's disease, and other diseases. In this study, we investigated the structure and redox properties of cross-linked amino acids and proteins produced by glyoxal. Model reactions between glyoxal or glycolaldehyde and the amino acids, alanine or N-alpha-acetyl-lysine, produced free radicals. The structure of this radical was identified by EPR spectroscopy as N- substituted pyrazinium radical cation, which was formed by cross-linking two amino acids. Glycation of BSA by these carbonyl compounds also generated stable protein-bound free radical species, probably the N-substituted pyrazinium radical cation as observed with amino acids. The glycated protein reduced ferricytochrome c to ferrocytochrome c, which was accompanied by a large increase in the EPR signal amplitude of the protein-bound free radical cation. In addition, the glycated protein catalyzed the oxidation of ascorbate. These results indicate that protein glycation generates active centers for catalyzing one electron redox reactions. One of the active centers generated by glyoxal is the cross-linked N-substituted pyrazine and its radical cation.