Vpr is an HIV-1 auxiliary regulatory protein packaged in the virion. It has been shown to enhance the nuclear transport of the HIV-1 pre-integration complex, activate transcription of cellular and viral promoters, and arrest the cell cycle at the G2/M check-point. We previously identified a cellular protein of 180 kDa (RIP) that interacted with HIV-1 Vpr specifically. We now rename this cellular protein as Vpr-binding protein, or VprBP. In this report, we describe the cloning of the VprBP cDNA that encodes 1507 aa residues and is identical to the previously cloned cDNA KIAA0800. We demonstrate that Vpr specifically interacts with recombinantly expressed VprBP in vitro as well as in vivo. Furthermore, Vpr interacts with the cellular endogenous VprBP in the context of the HIV-1 life cycle. Mutational analysis of VprBP suggests that the Vpr binding domain is located within the C-terminal half of VprBP, which has a Pro-rich domain and several Phe-x-x-Phe repeats. Subcellular fractionation studies show that both the endogenous VprBP and the adenovirus-expressed VprBP are distributed predominantly in the cytoplasmic fraction. Consistent with previous reports, the adenovirus-expressed Vpr is distributed in both the cytoplasmic and the nuclear fractions. However, when VprBP and Vpr are expressed together, Vpr is found almost exclusively in the cytoplasm. Expression of VprBP does not affect the nuclear transport of the adenoviral nuclear protein, pTP. VprBP expressed in insect cells also blocks the nuclear transport of a Vpr-GFP fusion protein, and VprBP mutants incapable of interacting with Vpr fail to block Vpr-GFP nuclear transport. We hypothesize that Vpr interaction with VprBP may cause changes in the host cell cytoplasm that affect HIV-1 pathogenesis as well as HIV-1 replication.