Methylglyoxal and other α-oxoaldehydes are formed from glycolytic intermediates and may be involved in the development of diabetic microangiopathy. Glyoxalase I and glyoxalase II metabolise methylglyoxal to d-lactic acid, via the intermediate S-d-lactoylglutathione. The activities of the glyoxalase enzymes and the concentrations of methylglyoxal and S-d-lactoylglutathione were measured in erythrocytes of 45 control and 85 diabetic subjects (41 with retinopathy and 44 uncomplicated). The concentration of S-d-lactoylglutathione was increased in diabetic patients vs. controls (21.4 ± 9.3 vs. 12.4 ± 4.8 μmol/l, P < 0.001), as was methylglyoxal (3.6 ± 2.3 vs. 1.4 ± 0.2 μmol/l, P < 0.001). There were no significant differences in the activities of glyoxalase I and glyoxalase II between diabetic patients and controls. For insulin-dependent patients only, those without retinopathy had a higher activity of glyoxalase II than those with retinopathy (P < 0.05). A group of age- and duration-matched insulin-dependent diabetic patients with retinopathy also had a higher activity of glyoxalase I compared with a group of diabetic patients without retinopathy (P < 0.025). This study provides evidence for elevated concentrations of oxoaldehydes in diabetes mellitus which might have pathogenic significance.