Congenitally acquired HIV infection may be uniquely suited to treatment via genetic engineering of CD34+ hematopoietic stem/progenitor cells. However, current technologies yield only a small percentage of mature cells that carry the inserted genes, and expression is frequently suppressed. Since clinical trials employing these methodologies have been proposed for anti-HIV gene therapy of HIV-infected children, we wished to assess, by in vitro modeling, the expected limits of transduction efficiency, expression, and antiviral activity using currently available methods. We measured retrovirus-mediated transduction in cord blood progenitors and their in vitro-derived progeny macrophages by Mo-MuLV vectors expressing a transdominant negative Rev (Rev(TD)). CFU-GM transduction efficiency ranged from 7 to 85%, with an average of 28%. Semiquantitative DNA PCR demonstrated ≤ 100 vector sequence copies per 1000 cells in monocyte/macrophage cultures, which were grown without selection to better model in vivo conditions. When challenged with the macrophage-tropic HIV-1(BaL) isolate, cultured macrophages from mock-transduced CFU-GM colonies supported infection in eight of eight experimental cultures, control LXSN-transduced progenitors supported infection in six of eight cultures, while macrophages derived from Rev(TD)-transduced CFU-GM colonies supported infection in four of eight cultures. Although these results support the ability of neo(r) retroviral vectors containing Rev(TD) to inhibit HIV replication, they indicate that further optimization of transduction efficiency and sustained expression will be required for effective anti-HIV protection in vivo.