Vpr is a virion-associated protein of human immunodeficiency type 1 (HIV-1) whose function in acquired immunodeficiency syndrome (AIDS) has been uncertain. Employing the yeast Saccharomyces cerevisiae as a model to examine the effects of HIV-1 auxiliary proteins on basic cellular functions, we found that the vpr gene caused cell growth arrest and structural defects indicated by osmotic sensitivity and gross cell enlargement. Production of various domains by gene expression showed that this effect arose from within the carboxyl-terminal third of the Vpr protein and implicated the sequence HFRIGCRHSRIG, containing two H(S/F)RIG motifs. Electroporation with a series of peptides containing these motifs caused structural defects in yeast that resulted in osmotic sensitivity. A protein with functions relating to the yeast cytoskeleton, Sac1p [Cleves, A. E., Novick, P.J. & Bankaitis, V.A. (1989) J. Cell Biol. 109, 2939-2950], shows sequence similarity to Vpr, and Vpr's effect in yeast may be to disrupt normal Sac1p functions. The Sac1p equivalent has not yet been described in mammalian cells, but in rhabdomyosarcoma and osteosarcoma cell lines Vpr also caused gross cell enlargement and replication arrest [Levy, D.N., Fernandes, L.S., Williams, W.V. & Weiner, D.B. (1993) Cell 72, 541-550]. We note that there is a correlation between the region containing the H(S/F)RIG motifs and the pathogenicity of primate lentiviruses and we suggest that the function of Vpr may be to bring about cell growth arrest and/or cytoskeletal changes as an early step in HIV-1 infection.