κB deregulation by HIV-1 are relevant for immune dysfunction in AIDS. We report that in single round HIV-1 infection, or single-pulse PMA stimulation, the HIV-1 Tat transactivator activated NF-κB by hijacking the inhibitor IκB-α and by preventing the repressor binding to the NF-κB complex. Moreover, Tat associated with the p65 subunit of NF-κB and increased the p65 DNA-binding affinity and transcriptional activity. The arginine- and cysteine-rich domains of Tat were required for IκB-α and p65 association, respectively, and for sustaining the NF-κB activity. Among an array of NF-κB-responsive genes, Tat mostly activated the MIP-1α expression in a p65-dependent manner, and bound to the MIP-1α NF-κB enhancer thus promoting the recruitment of p65 with displacemen