Methylglyoxal is a cytotoxic metabolite that is produced in vivo mainly from glycolysis. Increased production of methylglyoxal can be induced by tumor necrosis factor and occurs in a number of pathological conditions, including diabetes and neurodegenerative disorders. Methylglyoxal is highly reactive and can modify proteins, which results in the formation of advanced glycation end products. Yet, we, and others, have recently proposed a role for methylglyoxal as a signaling molecule. In this study, we show that methylglyoxal inhibits TNF-induced NF-kappaB activation and NF-kappaB-dependent reporter gene expression by inhibiting the DNA binding capacity of NF-kappaB p65. Methylglyoxal slightly delayed, but did not inhibit, TNF-induced degradation of IkappaBalpha and strongly inhibited TNF-induced NF-kappaB-dependent re-synthesis of IkappaBalpha. The TNF-induced nuclear translocation of NF-kappaB p65 was also delayed, but not inhibited, in the presence of methylglyoxal. TNF-induced phosphorylation of p65 was not affected by methylglyoxal. We show that the conserved Cys 38 residue, which is located in the DNA binding loop of NF-kappaB p65 and responsible for the redox regulation of the transcription factor, is involved in the methylglyoxal-mediated inhibition of p65 DNA-binding. Furthermore, overexpression of p65 inhibited TNF-induced cell death; however, in the presence of exogenously added methylglyoxal, overexpression of p65 caused far greater TNF-induced cell death. These findings suggest that methylglyoxal provides another control mechanism for modulating the expression of NF-kappaB-responsive genes and that methylglyoxal may be responsible for tipping the balance towards TNF-induced cell death in cells with constitutive NF-kappaB activation.