The viral infectivity factor gene (vif) of HIV-1 increases the infectivity of viral particles by inactivation of cellular anti-viral factors, and supports productive viral replication in primary human CD4 T cells and in certain non-permissive T cell lines. Here, we demonstrate that Vif also contributes to the arrest of HIV-1 infected cells in the G(2) phase of the cell cycle. Viruses deleted in Vif or Vpr induce less cell cycle arrest than wild-type virus, while cells infected with HIV-1 deleted in both Vif and Vpr have a cell cycle profile equivalent to that of uninfected cells. Furthermore, expression of Vif alone induces accumulation of cells in the G(2) phase of the cell cycle. These data demonstrate a novel role for Vif in cell cycle regulation and suggest that Vif and Vpr independently drive G(2) arrest in HIV-1 infected cells. Our results may have implications for the actions and interactions of key HIV-1 accessory proteins in AIDS pathogenesis.