Human immunodeficiency virus type 1 (HIV-1) Vpr participates in nuclear targeting of the viral preintegration complex in nondividing cells and induces G(2) cell cycle arrest in proliferating cells, which creates an intracellular milieu favorable for viral replication. Vpr also activates the transcription of several promoters and enhancers by a poorly understood mechanism. Vpr enhances glucocorticoid receptor (GR) signaling and may mediate the effects of steroids on HIV replication. More specifically, recombinant Vpr can potentiate virion production from U937 cells, downregulate NF-kappaB induction, and enhance programmed cell death, all effects also mediated by glucocorticoids. Vpr has been proposed to act as a GR coactivator, although other studies suggest that these enhancing effects are merely a consequence of G(2) cell cycle arrest. We now demonstrate that Vpr functions as a GR coactivator and that this activity is independent of cell cycle arrest. In addition, we show that the Vpr-induced coactivation requires an intact glucocorticoid response element, that it is dependent on the presence of hormone and the corresponding receptor, and that it is mediated by the two highly conserved leucine-rich domains within Vpr that resemble the GR coactivator signature motif.