The human immunodeficiency virus type 1 (HIV-1) Tat is a virally encoded protein that dramatically upregulates viral replication through interactions with the HIV-1 5' long terminal repeat (LTR) and cellular transcription factors. The HIV-1 LTR is divided into three major regions: modulatory, core and TAR. The modulatory region contains numerous cis-acting sequences for the binding of transcription factors including NF-kappaB, NF-AT, and AP-1. In several reports, Tat has been found to induce NF-kappaB activation of the HIV-1 LTR, while in other studies Tat has been reported to have no effect on activation of NF-kappaB. These discrepancies may arise from differences in experimental conditions such as the source of Tat (exogenous versus endogenous), the detection methods for NF-kappaB activation (DNA binding capability versus I kappaB degradation), and the types of reporters used (HIV-1 versus non-HIV-1 derived). To reconcile these differences we examined the effect of endogenous Tat on NF-kappaB activation, on I kappaB degradation and its interaction with upstream MAP3Ks. We demonstrate that although an 80% reduction in Tat-induced HIV-1 LTR activity can be detected if the kappaB binding sites are mutated, surprisingly endogenous Tat (expressed intracellularly by transfection) lacks direct effect on I kappaB degradation. Further analysis demonstrates that although Tat alone lacks direct effect on I kappaB alpha degradation or dissociation from NF-kappaB, Tat can substantially enhance the capacity of NF-kappaB-inducing kinase (NIK), but not MEKK1, to accelerate degradation of I kappaB. We propose a model to explain these collective experimental findings.