Lentiviral accessory proteins are thought to play important roles in regulating the viral replication through modulation of host cell functions. For example, Vpr of human immunodeficiency virus type 1 (HIV-1) induces the cell cycle G2 arrest in a host cell-specific manner. Similarly, HIV-2 Vpr, but not Vpx, has been shown to induce G2 arrest in primate cells. It has also been reported that Orf-A of feline immunodeficiency virus (FIV) induces G2 arrest in a simian cell line. However, activities of these non-HIV-1 accessory proteins in different cellular context are unclear. In this study, effects of HIV-2 Vpr, Vpx and FIV Orf-A on cell cycle progression were compared with those of HIV-1 Vpr in various mammalian cell lines and the fission yeast. These non-HIV-1 accessory proteins induced the cell cycle arrest in a host cell-specific manner, and their specificities were different from each other. Interestingly, HIV-2 Vpx-induced G2 arrest in bovine MDBK cells. It was also notable that HIV-2 Vpx and FIV Orf-A appeared to block the cell separation in the fission yeast. The host cell-specific activities of different lentiviral accessory proteins revealed in this study may provide a useful basis for elucidating the mechanism of their functions.