To attain a profile of protein expression during diabetes, we applied proteomic analysis to glomeruli of 160 day old db/db diabetic and db/m non-diabetic mice. Glomerular proteins were extracted and separated by two-dimensional gel electrophoresis to construct a proteome map. One hundred and ninety proteins were identified by matrix-assisted laser desorption and ionization-time of flight mass spectrometry and peptide mass fingerprinting. Out of 105 analyzed spots, expression of 40 proteins was increased with diabetes, including, the anti-oxidative enzymes peroxiredoxin 1 and 3, glutathione peroxidase 1, and superoxide dismutase 1, suggesting an adaptive response to oxidative stress associated with this diabetic model. However, activity of glutathione peroxidase and superoxide dismutase was unaltered in glomeruli of diabetic mice. Expression of glyoxalase I was increased in glomeruli of diabetic mice. Because the co-factor for glyoxalase I, glutathione, is decreased in the renal cortex of db/db mice, renal cortical glyoxalase I activity was measured, in vitro, with fixed amounts of exogenous glutathione. Glyoxalase I activity was decreased in the renal cortex of db/db mice. These data indicate that diabetes-induced decreases in glyoxalase activity are likely to be due to glutathione-dependent and independent mechanisms and that increased expression of glyoxalase I may represent an insufficient adaptive response to increased methylglyoxal formation.