We have previously established a stable HIV-1 packaging cell line, psi 422, which yielded high titers of an HIV-1 vector capable of efficiently transducing CD4+ cells. In order to increase the safety of this gene delivery system, we have now replaced the HIV-1 vector with an HIV-2 vector to abolish any risk of homologous recombination between the packaging cells and the vector. The HIV-2 vector was also modified by insertion of a cis-acting constitutive transport element which confers Rev independence. The supernatant of psi 422 cells stably transfected with this new vector was capable of transducing CD4+ cells with a titer of 10(4) transducing units per milliliter. This result shows that cross-packaging of HIV-2 vectors with the HIV-1 packaging cells is quite efficient. Using this new stable HIV-1/HIV-2 gene delivery system, we were able to transduce human monocyte-derived primary macrophages, which are refractory to murine retrovirus-mediated transduction. The availability of a stable HIV-based gene delivery system for macrophages, a key target cell in HIV infection; is an important advance in gene therapy for AIDS.