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HIV-1 Vpr is a 96-amino acid auxiliary protein that performs numerous activities during viral infection. In the present study, 10 antibodies were generated after mice immunization with either the N- or the C-terminus domain of Vpr, respectively, Vpr(1–51) and Vpr(52–96). ELISA and immunoblot experiments using pure synthetic overlapping Vpr peptides suggested that these anti-Vpr antibodies could be classified into five groups and that they recognized conformational or linear Vpr epitopes. Further analysis revealed the effect of C-terminal arginine mutations on the antibody binding. Two of the antibodies precipitated Vpr expressed after transfection of a Vpr-encoding vector in human cells. More importantly, one of them was able to detect Vpr in HIV-1-infected U1 cells and in HIV-1-infected human PBMC. Surface plasmon resonance experiments demonstrated that some of these antibodies prevented the interaction between Vpr and one of its cellular partners, the adenine nucleotide translocator. Thus, these anti-Vpr monoclonal antibodies may be useful to any laboratory working on the molecular mechanism of HIV-1 infection.