In unstimulated cells, transcription factor NF-kappaB is retained in the cytoplasm by interaction with the inhibitory protein, IkappaBalpha. Appropriate cellular stimuli inactivate IkappaBalpha by phosphorylation, ubiquination, and proteolytic degradation, which allows NF-kappaB to translocate to the nucleus and modulate gene expression. 4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, inhibits activation of NF-kappaB in the human colorectal carcinoma cell line (RKO) and human lung carcinoma cell line (H1299). Pretreatment of cells with HNE dose-dependently suppresses tetradecanoylphorbol acetate (TPA)/ionomycin (IM)-induced NF-kappaB DNA binding activity and transactivation of luciferase-based reporter constructs. HNE pretreatment has no affect on TPA/IM-induced AP-1 DNA binding activity. HNE inhibits TPA/IM-induced degradation of IkappaBalpha in both H1299 and Jurkat T cells. The accumulation of IkappaBalpha parallels the inhibition of its phosphorylation. At doses that inhibit IkappaBalpha degradation, HNE inhibits IkappaB kinase (IKK) activity by direct reaction with IKK. Covalent adducts of HNE to IKK are detected on Western blots using antibodies against IKK or HNE-protein conjugates. Addition of dithiothreitol prevents HNE modification of IKK. Thus, HNE is an endogenous inhibitor of NF-kappaB activation that acts by preventing IKK activation and subsequent IkappaBalpha degradation.