The effect of methylglyoxal (MG) on the aerobic glycolysis of Ehrlich ascites carcinoma (EAC) cells has been tested. Methylglyoxal inhibited glucose utilization and glucose 6-phosphate (G6P) and L-lactate formation in whole EAC cells. Methylglyoxal strongly inactivated glyceraldehyde 3-phosphate dehydrogenase (GA3PD) of the malignant cells, whereas MG has little inactivating effect on this enzyme from several normal sources. Methylglyoxal also inactivated only the particulate hexominase of the EAC cells, but this inactivation was less pronounced than the effect on GA3PD. Methylglyoxal has little inactivating effect on glucose 6-phosphate dehydrogenase (G6PD), and no effect on L-lactate dehydrogenase (LDH) of the malignant cells. Glucose-dependent L-lactic acid formation of EAC-cell-free homogenate was strongly inhibited by MG, but when GA3PD of normal cells was added to this homogenate, significant lactate formation was observed even in the presence of MG. Methylglyoxal also inhibited the respiration of EAC-cell mitochondria. Respiration of mitochondria isolated from liver and kidney of normal mice, however, remained unaffected. As a consequence of the inhibition of glycolysis and mitochondrial respiration, the ATP level of the EAC cells was drastically reduced. Studies reported herein strongly suggest that the tumoricidal effect of MG is mediated at least in part through the inhibition of mitochondrial respiration and inactivation of GA3PD, and this enzyme may play an important role in the high glycolytic capacity of the malignant cells.