Methylglyoxal (MGO) can modify tissue proteins through the Maillard reaction, resulting in advanced glycation end products (AGEs), which can alter protein structure and functions. Several MGO-derived AGEs have been described, including argpyrimidine, a fluorescent product of the MGO reaction with arginine residues. We detected significant amount of argpyrimidine in rat kidney mesangial cells cultured in media containing high concentrations of glucose. Heat shock protein 27 (Hsp27) was identified by liquid chromatography tandem mass spectrometry as a major anti-argpyrimidine immunoreactive protein. We confirmed this finding by reciprocal co-immunoprecipitation and by Western analysis. Diabetic rats contained more argpyrimidine-modified glomerular Hsp27 than non-diabetic animals. Additional studies showed that MGO-induced modification of Hsp27 decreased its binding to cytochrome c. Our results suggest that Hsp27 is a major target for MGO modification in mesangial cells.