Two cytokine-inducible kinases, IKKα and IKKβ, are components of a 700-kDa kinase complex that specifically phosphorylates IκB. Phosphorylation of IκB by IKK leads to its ubiquitination and subsequent degradation, resulting in the nuclear translocation of NF-κB. The oncogenic protein Tax, encoded by human T-cell leukemia virus type-1 (HTLV-1), stimulates IKK activity to result in constitutive nuclear levels of NF-κB. In an attempt to gain insights into the mechanism by which Tax mediates constitutive activation of the NF-κB pathway, we analyzed the chromatographic distribution of IKK proteins using cellular extracts prepared from three T lymphocytes either lacking or containing Tax. IKK kinase activity and the distribution of proteins in the IKK complex were characterized. In extracts prepared from cells containing Tax, the activity of both IKKα and IKKβ present in the 700-kDa IKK complex were increased. Surprisingly, cell lines expressing Tax also contained an additional peak of IKKβ, but not IKKα activity, that migrated at 300 kDa rather than at 700 kDa. We noted that extracts containing Tax had extremely low levels of IκBβ, but not IκBα, and contained predominantly a truncated form of the MAP3K MEKK1. These results suggest that Tax may target several components of the NF-κB pathway leading to constitutive activation of this important regulator of cellular gene expression.