The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr appears to make a substantial contribution to the replication of HIV-1 in established T cell lines when HIV-1 is present at very low multiplicities of infection. However, the role of Vpr in viral replication in primary CD4 + T cells remains to be clarified. In this study, we generated a panel of viruses that encoded mutant forms of Vpr that lacked either the ability to accumulate in the nucleus and induce G 2 arrest or the ability to induce apoptosis, which has been shown to occur independently of G 2 arrest of the cell cycle. We demonstrate here that the nuclear localization of Vpr and consequent G 2 arrest but not the induction of apoptosis by Vpr are important for viral replication in primary CD4 + T cells at both high and low multiplicities of infection. Viruses that encoded mutant forms of Vpr that failed to be imported into the nucleus in the presence of cytoplasmic extracts from primary CD4 + T cells in an in vitro nuclear import assay replicated at drastically reduced rates. Thus, Vpr might be a key regulator of the viral nuclear import process during infection in primary CD4 + T cells. By contrast, a mutant form of Vpr that exhibited diffuse cytosolic staining exclusively in an immunofluorescence assay of HeLa cells and was not imported into nucleus by the cytosol from HeLa cells was effectively imported into the nucleus by cytosol from primary CD4 + T cells. This Vpr mutant virus replicated well in primary CD4 + T cells, indicating that cellular factors in primary CD4 + T cells are indispensable for the accumulation of Vpr in the nucleus and, thus, for viral replication. Our results suggest that the nuclear import of Vpr might be a good target in efforts to block the early stages of replication of HIV-1. © 2004 Elsevier Inc. All rights reserved.