Nuclear factor-κB (NF-κB)/Rel transcription factors regulate genes that control cell proliferation, survival, and transformation. In normal breast epithelial cells, NF-κB/Rel proteins are mainly sequestered in the cytoplasm bound to one of the specific inhibitory IκB proteins, whereas in breast cancers they are activated aberrantly. Human breast tumor cell lines, carcinogen-transformed mammary epithelial cells, and the majority of primary human or rodent breast tumor tissue samples express constitutively high levels of nuclear NF-κB/Rel. To begin to understand the mechanism of this aberrant NF-κB/Rel expression, in this study we measured the activity of the major kinases implicated in regulation of IκB stability, namely IKKα, IKKβ, and protein kinase, CK2 (formerly casein kinase II). Hs578T, D3-1, and BP-1 breast cancer cell lines displayed higher levels of IKKα, IKKβ, and CK2 activity than untransformed MCF-10F mammary epithelial cells. Inhibition of IKK activity upon expression of dominant negative kinases or of CK2 activity by treatment with selective inhibitors decreased NF-κB/Rel activity in breast cancer cells. Inactivation of the IκB kinase complex in Hs578T cells via expression of a dominant negative IKKγ/NF-κB essential modulator/IKK-associated protein 1 reduced soft agar colony growth. Thus, the aberrant expression of CK2 or IKK kinases promotes increased nuclear levels of NF-κB/Rel and transformation of breast cancer cells. Furthermore, primary human breast cancer specimens that displayed aberrant constitutive expression of NF-κB/Rel were found to exhibit increased CK2 and/or IKK kinase activity. These observations suggest these kinases play a similar role in an intracellular signaling pathway that leads to the elevated NF-κB/Rel levels seen in primary human mammary tumors and, therefore, represent potential therapeutic targets in the treatment of patients with breast cancer.