HIV-1 vpr encodes a 96-amino acid, nuclear protein whose function is not well understood. Unlike the other lentivirus regulatory proteins, Vpr is present in virions at relatively high copy number. In cells, Vpr is localized to the nucleus. Possible functions for vpr consistent with these findings include the nuclear import of preintegration complexes, transactivation of cellular genes, or induction of cellular differentiation. We show here, using both replication competent, macrophage-tropic virus and a sensitive, single-cycle luciferase HIV-1 reporter vector, that vpr is important for efficient viral replication in primary monocyte/macrophages, but appears to play no role in activated or resting T cell infection. The block to infection in monocytes was localized by PCR analysis of newly synthesized viral DNA and with the luciferase reporter vector to a stage in the viral life cycle after entry and reverse transcription, yet prior to, or at the time of, proviral transcription. In addition, infection of mononuclear phagocytes with virions that had been loaded with Vpr molecules in the producer cells by trans-complementation still showed a vpr-phenotype. These data suggest a role for vpr molecules produced in newly infected cells, in addition to its presumed function in the virion.