Vpr, one of the accessory gene products of HIV-1, is a 96-residue protein with several functions. It is involved in import of the HIV-1 preintegration complex into the nucleus of nondividing cells, in cellular differentiation, inducing cell cycle arrest at the G2/M phase, in immune suppression, and in enhancement of replication of the virus. We found recently that Vpr interferes with the proliferation of mouse NIH3T3 fibroblasts but fails to arrest these cells in the G2 phase. Thus, it seems possible that Vpr might retard cell proliferation via a novel pathway that is distinct from G2 arrest. To elucidate the mechanism by which Vpr induces the retardation of cell growth, we developed a panel of expression vectors that encoded Vpr molecules with deletions of specific putative domains, namely, the first α-helical domain, the second α-helical domain, a leucine zipper- like domain, and an arginine-rich carboxy-terminal domain. These vectors were introduced into HeLa cells since expression of Vpr can induce G2 arrest in such cells. A carboxy-terminally truncated form of Vpr, C81, which failed to induce G2 arrest, led to the G1 arrest and retained the ability to prevent cell proliferation. All the other mutant proteins had completely lost the capacity to induce G2 arrest and to suppress growth. Substitutions of Ile/Leu for Pro at positions 60, 67, 74, and 81 within the leucine zipper- like domain of Vpr or of C81 revealed that Ile60, Leu67, and Ile74 play an important role in the C81-induced suppression of growth, while Ile74 and Ile81 were found to be indispensable for Vpr-induced G2 arrest. Collectively, our results strongly suggest that Vpr can retard cell proliferation independently of G2 arrest of the cell cycle.