The Vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 14-kDa protein that prevents cell proliferation by causing arrest in the G2/M phase of the cell cycle. Here we report the first evidence that Vpr activates the expression and transcription of the cyclin-dependent kinase inhibitor p21/Waf1/Cip1 (hereafter p21), an inhibitor of the G1 and G2/M phase transitions in T lymphoid and myeloid cells. Vpr activated p21 protein expression in a dose-dependent manner. Vpr also caused a three- to eightfold induction of the p21 promoter. This induction was dose- and time-dependent and was comparable to levels of p21 induction induced by p53. Of note, Vpr activated p21 transcription in endogenous p53 positive cells, but not in p53-deleted or p53 nonfunctional cells. Vpr and p53 had an additive effect on p21 transcription. Mutational analysis indicated that wt Vpr, but not cell cycle inactive Vpr mutants, activated the p21 promoter. These data demonstrate that HIV-1 Vpr utilizes the cyclin-dependent kinase inhibitor p21, in addition to cdc2, to arrest cells in G2/M.