Methylglyoxal (MG) contributes significantly to the carbonyl stress in uremia; however, the reason for its increased concentration is not clear. Thus, the present study was aimed to investigate the formation and degradation of MG in the erythrocytes of hemodialyzed (HD) patients with end-stage renal disease. In 22 nondiabetic patients on long-term HD, erythrocyte MG and d-lactate levels, glyoxalase activities, and whole blood reduced glutathione content were determined. The data were compared with those from 22 healthy controls. Erythrocyte MG and d-lactate production were also investigated in vitro under normoglycemic (5 mmol/L) and hyperglycemic (50 mmol/L) conditions. The erythrocyte MG levels were elevated (P < .001) in the HD patients. The blood reduced glutathione content and glyoxalase I activity were similar to the control levels, but the glyoxalase II activity was significantly (P < .005) increased. In the normoglycemic in vitro model, production of both MG (P < .001) and d-lactate (P < .002) was significantly enhanced in the HD erythrocytes relative to the controls. During hyperglycemia, the MG formation and degradation rates were further increased (P < .001). The present study demonstrated an increased formation of MG in the erythrocytes of HD patients. This seemed to be related to a glucose metabolism disturbance of the cells. The degradation system of MG was also activated; still, it was not able to counteract the high rate of MG formation. The alterations and imbalance of these metabolic processes may contribute to the carbonyl overload and stress in the HD patients.