The nef genes of human and simian immunodeficiency viruses code for a membrane associated protein critical for AIDS development. SIVmac Nef presents C-terminal a 27 amino acid extension absent of HIV-1 Nef. To estimate the influence of this C-terminal domain on virus properties, we constructed viruses derived from SIVmac239 by replacing SIV nef with HIV-1 Lai nef gene (SHIV NefLai4) or with a sequence encoding a Nef fusion protein: HIV-1 Lai Nef/SIV Nef-Cterm (SHIV-Cterm). The recombinant viruses replicated efficiently in vitro in CEMx174 cells and in activated macaque PBMCs. The addition of SIV Nef C-terminal domain to HIV-1 Nef in SHIVNefLai4 did not change the in vitro properties of the chimeric virus, both viruses being more infectious than a nef deleted virus. Although the half-life of Nef fusion protein was augmented, SHIV-Cterm remained slightly less infectious than SIVmac239.