To investigate the function of vpx, a gene in HIV-2 and SIV, but not in HIV-1, three site-directed mutants (pMX) were constructed from a functional proviral HIV-2 plasmid clone (pSE). Transfection of COS-1 cells with all three mutants as well as pSE gave rise to equivalent amounts of virus. Each virus could be passaged in H9 and CEM lymphoid cell lines, peripheral blood lymphocytes, and monocytes with equal efficiency and demonstrated similar cytopathic effects. Hybridization data with DNA from the infected cells demonstrated the presence of similar levels of viral sequences and the mutations in each of the MX-infected cell lines. Immunoprecipitation analysis demonstrated a 16-kDa VPX protein in cells infected with SE virus, as well as in the virus particles, but not in cells infected with MX viruses or the particles themselves. However, equivalent levels of gag and env proteins were demonstrated in all infected cells and virion preparations. These data suggest that VPX is dispensable for virus replication and cytopathicity.