Oxidative modification of proteins has been implicated in aging, ischemia reperfusion, carcinogenesis, and other phenomena. Oxidation of the C-2 position of the imidazole ring of histidine converts the residue to 2-oxohistidine, a novel amino acid which may serve as a marker of oxidative modification of proteins (K. Uchida and S. Kawakishi,J. Biol. Chem.269, 2405–2410, 1994). It has been identified in oxidatively modified proteins by electrochemical detection during reverse-phase high-pressure liquid chromatography, by mass spectrometry, and as the phenylthiohydantoin after Edman degradation, but not by amino acid analysis of protein hydrolysates. We now describe procedures for stabilizing 2-oxohistidine which allow its quantification by routine methods of amino acid analysis. These include classical ion exchange chromatography with postcolumn derivatization byo-phthaldialdehyde, reverse-phase chromatography with precolumn derivatization byo-phthaldialdehyde, and reverse-phase chromatography with precolumn derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Using these techniques, a previously unidentified amino acid which appears during the oxidative inactivation of glutamine synthetase was shown to be 2-oxohistidine. One picomole of 2-oxohistidine was readily detected in a protein hydrolysate containing 1700 pmol total amino acids.