The functional activity of SIVmac251 Rev was altered by introducing amino acid changes inside and chain termination mutations after the Rev response element-binding region (RBR) of the protein. The effects of specific mutations were evaluated by transfecting proviral DNAs into the HeLa cell line and into HeLa cells constitutively expressing either HIV-1 Rev or HTLV-1 Rex proteins. Cell-free supernatants from these transient expression assays were further characterized by infecting CD4-positive lymphoid cell lines H9 and MT-4, the latter abortively infected with HTLV-1, and human peripheral blood mononuclear cells. These results together with the data from cotransfection experiments show that SIV can be attenuated up to 95% by introducing changes into the arginine-rich domain, RBR, of Rev. These recessive mutations were efficiently complemented in trans by HIV-1 Rev, SIV Rev, and HTLV-I Rex proteins. In contrast, the mutants of Rev protein that had a chain termination after RBR were trans-dominant negative and could not be trans-complemented with any of these three regulatory proteins. When additional mutations were inserted into the RBR of these trans-dominantly negative Rev proteins, complementation was obtained again.