Glyoxal is a reactive alpha-oxoaldehyde that is a physiological metabolite formed by lipid peroxidation, ascorbate autoxidation, oxidative degradation of glucose and degradation of glycated proteins. Glyoxal is capable of inducing cellular damage, like methylglyoxal (MG), but may also accelerate the rate of glycation leading to the formation of advanced glycation end-products (AGEs). However, the mechanism of glyoxal cytotoxicity has not been precisely defined. In this study we have focused on the cytotoxic effects of glyoxal and its ability to overcome cellular resistance to oxidative stress. Isolated rat hepatocytes were incubated with different concentrations of glyoxal. Glyoxal by itself was cytotoxic at 5mM, depleted GSH, formed reactive oxygen species (ROS) and collapsed the mitochondrial membrane potential. Glyoxal also induced lipid peroxidation and formaldehyde formation. Glycolytic substrates, e.g. fructose, sorbitol and xylitol inhibited glyoxal-induced cytotoxicity and prevented the decrease in mitochondrial membrane potential suggesting that mitochondrial toxicity contributed to the cytotoxic mechanism. Glyoxal cytotoxicity was prevented by the glyoxal traps d-penicillamine or aminoguanidine or ROS scavengers were also cytoprotective even when added some time after glyoxal suggesting that oxidative stress contributed to the glyoxal cytotoxic mechanism.