Interplay of the HIV-1 regulatory protein, Tat, with several cellular factors plays an important role in transcriptional regulation of the viral promoter, the long terminal repeat (LTR). Special attention has been paid to NF-κB, a family of inducible transcription factors, which interact with a specific DNA motif within the LTR. Here, we report on the physical and functional interaction of NFBP, a recently identified protein that interacts with the P65 subunit of NF-κB, with HIV-1 Tat. NFBP colocalizes with Tat in the nucleus and nucleoli, recognizes the amino acid residues 37 to 48 of Tat, and its interaction is modulated by RNA molecules. The interaction of NFBP with Tat modulates the synergism between Tat and P65 in activating LTR transcription. In the absence of the κB-binding sites, NFBP augments the TAR-dependent activation by Tat, yet it interferes with the synergistic effect of P65 and Tat on LTR transcription. © 2005 Wiley-Liss, Inc.