Activation of the NF-kappaB/Rel family of transcription factors proceeds through a catalytic complex containing IkappaB kinase (IKK)-1 and IKK2. Targeted disruption of each of the IKK genes suggests that these two kinases may mediate distinct functions in the activation pathway. In our studies of the human T-cell lymphotropic virus type 1 (HTLV-1) Tax oncoprotein, we have uncovered a new function of IKK1 required for complete activation of the NF-kappaB transcriptional program. In IKK1(-/-) murine embryonic fibroblasts (MEFs), Tax normally induced early NF-kappaB activation events. However, NF-kappaB induced by Tax in these IKK1(-/-) cells was functionally impaired. In IKK1(-/-) (but not wild-type) MEFs, Tax failed to activate several different kappaB reporter constructs or to induce the endogenous IkappaBalpha gene. In contrast, Tax normally activated the cAMP-responsive element-binding protein/activating transcription factor pathway, leading to full stimulation of an HTLV-1 long terminal repeat reporter construct in IKK1(-/-) cells. Furthermore, reconstitution of IKK1(-/-) cells with kinase-proficient (but not kinase-deficient) forms of IKK1 restored the Tax induction of full NF-kappaB transactivation. We further found that the defect in NF-kappaB action in IKK1(-/-) cells correlated with a failure of Tax to induce phosphorylation of the RelA/p65 subunit of NF-kappaB at Ser(529) and Ser(536). Such phosphorylation of RelA/p65 was readily detected in wild-type MEFs. Phosphorylation of Ser(536) was required for a complete response to Tax expression, whereas phosphorylation of Ser(529) appeared to be less critical. Together, these findings highlight distinct roles for the IKK1 and IKK2 kinases in the activation of NF-kappaB in response to HTLV-1 Tax. IKK2 plays a dominant role in signaling for IkappaBalpha degradation, whereas IKK1 appears to play an important role in enhancing the transcriptional activity of NF-kappaB by promoting RelA/p65 phosphorylation.