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Nuclear factor kappaB (NF-kappaB) plays an important role in regulating cellular transformation and apoptosis. The human T-cell lymphotropic virus type I protein, Tax, which is critical for viral transformation, modulates the transcription of several cellular genes through activation of NF-kappaB. We have demonstrated previously that Tax inhibits p53 activity through the p65/RelA subunit of NF-kappaB. We now present evidence that suggests that the upstream kinase IKKbeta plays an important role in Tax-induced p53 inhibition through phosphorylation of p65/RelA at Ser-536. First, mouse embryo fibroblast (MEF) IKKbeta-/-cells did not support Tax-mediated p53 inhibition, whereas MEFs lacking IKKalpha allowed Tax inhibition of p53. Second, transfection of IKKbeta wild type (WT), but not a kinase-dead mutant, into IKKbeta-/-cells rescued p53 inhibition by Tax. Third, the IKKbeta-specific inhibitor SC-514 decreased the ability of Tax to inhibit p53. Fourth, we show that phosphorylation of p65/RelA at Ser-536 is important for Tax inhibition of p53 using MEF p65/RelA-/-cells transfected with p65/RelA WT or mutant plasmids. Moreover, Tax induced p65/RelA Ser-536 phosphorylation in WT or IKKalpha-/- cells but failed to induce the phosphorylation of p65/RelA Ser-536 in IKKbeta-/-cells, suggesting a link between IKKbeta and p65/RelA phosphorylation. Consistent with this observation, blocking IKKbeta kinase activity by SC-514 decreases the phosphorylation of p65/RelA at Ser-536 in the presence of Tax in human T-cell lymphotropic virus type I-transformed cells. Finally, the ability of Tax to inhibit p53 is distinguished from the NF-kappaB transcription activation pathway. Our work, therefore, describes a novel Tax-NF-kappaB p65/RelA pathway that functions to inhibit p53 but does not require NF-kappaB transcription activity.