We have previously described the cloning and sequencing of a novel stain of human immunodeficiency virus type 2 (HIV-2) called HIV-2NIH-Z. A plasmid clone, pHIV2Z, containing the full-length provirus has now been constructed, and virus particles have been obtained upon transfection into COS-1 and H-9 cells. These particles can infect a number of T-cell lines and exert a cytopathic effect on fresh human and macaque peripheral blood lymphocytes. The cloned virus is biologically and morphologically indistinguishable from its parental uncloned strain as shown by restriction enzyme analysis, electron microscopy, and kinetics of infection. However, as shown by radioimmunoprecipitation assays, the cloned virus-infected cells express a full-length gp41 protein as predicted by the nucleotide sequence, whereas the wild-type parental strain expresses a truncated gp33 protein. Both the parental strain and the cloned virus possess a deletion encompassing the end of the nef gene within the U3 region which apparently does not affect their in vitro cytopathic and replicative capacities.