Protein carbonyls are reported to increase in aging and in pathologies such as Alzheimer's disease and ischemic injury. Detailed study of this important issue has, however, been hampered by lack of an appropriate method to identify individual carbonylated proteins. We describe here an immunoblot method to investigate protein carbonyls reactive to 2,4-dinitrophenyl hydrazine. Rabbit polyclonal antibodies against 2,4-dinitrophenyl hydrazine were used to study the proteins derivatized by the reagent in one- or two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting. More than 25 proteins with high carbonyl contents were clearly demonstrated in two-dimensional immunoblot of rat tissue soluble proteins. The method could detect concentrations as low as 1 pmol of carbonyls. The signals were mostly abolished by prior treatment of tissue proteins with sodium borohydride to reduce carbonyls. Fragments generated by V8 protease digestion of a single protein exhibited signal intensities of varying degrees, indicating that carbonyla-tion is not uniform in different amino acid sequences. Proteins treated with glucose or aldehydes gave rise to positive signals, suggesting that the finding of carbonyls in tissue proteins is not necessarily an indication of direct oxidation of side chains of amino acid residues.