The Vpr gene of human immunodeficiency virus type 1 and type 2 (HIV-1, HIV-2) and simian immunodeficiency virus (SIV) encodes a small nuclear protein which is virion-associated and assists nuclear transport of the preintegration complex. Expression of HIV-1 Vpr has been shown to induce differentiation and prevent proliferation of human cells. HIV-1 Vpr has also been shown to arrest cell growth and cause morphological defects in yeast. In contrast, the Vpx gene of HIV-2 and SIV, which shares sequence homology with Vpr, does not seem to inhibit proliferation of human cells. It has been suggested that the cell cycle arrest effect of Vpr and Vpx is species and cell-type dependent. In this study, we have taken advantage of a conditional expression system to characterize the growth inhibitory effects of Vpr and Vpx of HIV-1, HIV-2, and SIV in the fission yeast Schizosaccharomyces pombe. Our results show that both Vpr and/or Vpx of HIV-1, HIV-2, and SIV arrest cell growth in S. pombe, and HIV-1 Vpr is more cytotoxic than HIV-2 or SIV Vpr or Vpx. Flow cytometry analysis indicated that yeast cells cease proliferating with DNA contents indicative of arrest in G1 and G2, with some cells showing signs of overreplication of DNA. While the observed cell cycle arrest phenotype was not identical to that observed in mammalian cells, there were similarities of growth arrest phenotype caused by Vpr and Vpx in yeast and mammalian cells. Specifically, the observation that yeast and mammalians cell both arrest in G2 with reduced p34/cdc2 kinase activity indicates that Vpr and Vpx interact with conserved target(s) in yeast and mammalian cells. The ability to use genetic analysis to elucidate the mechanisms involved makes S. pombe an excellent model system in which to study the effects of Vpr and Vpx on cellular function.