Ethyl pyruvate has been shown to have anti-inflammatory properties in numerous cell culture and animal studies. In this series of experiments, we tested the hypothesis that ethyl pyruvate inhibits signaling by the pro-inflammatory transcription factor, NF-kappaB. Ethyl pyruvate inhibited luciferase expression in lipopolysaccharide-stimulated murine macrophage-like RAW 264.7 cells transfected with an NF-kappaB-dependent luciferase reporter vector. Ethyl pyruvate also decreased NF-kappaB DNA-binding activity in lipopolysaccharide-stimulated RAW 264.7 cells and decreased lipopolysaccharide-induced expression of an NF-kappaB-dependent gene, inducible nitric oxide synthase. Ethyl pyruvate had no effect on the degradation of IkappaBalpha or IkappaBbeta in lipopolysaccharide-stimulated RAW 264.7 cells, suggesting that ethyl pyruvate acts distally to this step in the activation of NF-kappaB. In a cell-free system, binding of p50 homodimers to an NF-kappaB consensus oligonucleotide sequence was unaffected by ethyl pyruvate over a wide range of concentrations, indicating that ethyl pyruvate probably does not modify or interact with the p50 subunit of NF-kappaB. In contrast, ethyl pyruvate inhibited DNA binding by ectopically overexpressed wild-type p65 homodimers. However, ethyl pyruvate failed to inhibit the DNA-binding activity of homodimers of an overexpressed mutant form of a p65 with substitution of serine for cysteine 38. Taken together, these results suggest that ethyl pyruvate inhibits DNA-binding by covalently modifying p65 at Cys(38). We conclude that some of the beneficial anti-inflammatory effects of ethyl pyruvate may be due to modification of p65, thereby inhibiting signaling via the NF-kappaB pathway.