We showed previously that the signal transcription factor nuclear factor-kappaB (NF-kappaB) is aberrantly activated and that inhibition of NF-kappaB induces cell death and inhibits tumorigenesis in head and neck squamous cell carcinomas (HNSCC). Thus, identification of specific kinases underlying the activation of NF-kappaB could provide targets for selective therapy. Inhibitor-kappaB (IkappaB) kinase (IKK) is known to activate NF-kappaB by inducing NH(2)-terminal phosphorylation and degradation of its endogenous inhibitor, IkappaB. Casein kinase 2 (CK2) was previously reported to be overexpressed in HNSCC cells and to be a COOH-terminal IKK, but its relationship to NF-kappaB activation in HNSCC cells is unknown. In this study, we examined the contribution of IKK and CK2 in the regulation of NF-kappaB in HNSCC in vitro. NF-kappaB activation was specifically inhibited by kinase-dead mutants of the IKK1 and IKK2 subunits or small interfering RNA targeting the beta subunit of CK2. CK2 and IKK kinase activity, as well as NF-kappaB transcriptional activity, was shown to be serum responsive, indicating that these kinases mediate aberrant activation of NF-kappaB in response to serum factor(s) in vitro. Recombinant CK2alpha was shown to phosphorylate recombinant IKK2 as well as to promote immunoprecipitated IKK complex from HNSCC to phosphorylate the NH(2)-terminal S32/S36 of IkappaBalpha. We conclude that the aberrant NF-kappaB activity in HNSCC cells in response to serum is partially through a novel mechanism involving CK2-mediated activation of IKK2, making these kinases candidates for selective therapy to target the NF-kappaB pathway in HNSCC.