Overexpression of epidermal growth factor (EGF) receptor and constitutive activation of nuclear factor-kappaB (NF-kappaB) are frequently encountered in tumor cells. Although EGF has been shown to induce NF-kappaB activation, the mechanism is poorly understood. EGF activated NF-kappaB DNA binding, induced NF-kappaB reporter activity and the expression of antiapoptotic and cell-proliferative gene products. Interestingly, non-small cell lung adenocarcinoma cell lines (HCC827 and H3255), which exhibit EGFR amplification, showed ligand-independent activation of NF-kappaB. Unlike tumor-necrosis factor (TNF), however, EGF failed to induce IkappaBalpha phosphorylation and ubiquitination and the activation of IkappaBalpha kinase (IKK). Although DN-IKKbeta inhibited TNF-induced NF-kappaB activity, DN-IKKbeta had no effect on EGF-induced NF-kappaB activation, suggesting that EGF-induced NF-kappaB activation is IKK independent. Using dominant-negative plasmids, we also demonstrated the role of TRADD, TRAF2, NIK and Ras in EGF-induced NF-kappaB activation. By using specific antibodies and IkappaBalpha plasmid, which is mutated at tyrosine 42 to phenylalanine, we show that EGF induced the tyrosine phosphorylation of IkappaBalpha at residue 42. Furthermore, EGF receptor kinase inhibitor blocked IkappaBalpha phosphorylation and consequent NF-kappaB activation. Overall, our results indicate that tyrosine phosphorylation of IkappaBalpha at residue 42 is critical for EGF-induced NF-kappaB activation pathway.