Gene delivery via the hematopoietic stem cell (HSC) offers an attractive means to introduce antiviral genes into both T cells and macrophages for acquired immunodeficiency syndrome (AIDS) gene therapy. An amphotropic retroviral vector encoding a bicistronic gene coexpressing RevM10 and the murine CD8alpha' chain (lyt2) was developed to transduce HSC/progenitor cells. After transduction of CD34+ cells isolated from human umbilical cord blood, the lyt2 molecule detected by flow cytometry was used to monitor the level of gene transduction and expression and to enrich RevM10-expressing cells by cell sorting without drug selection. Using this quantitative method, high levels of gene transduction and expression (around 20%) were achieved by high-speed centrifugation of CD34+ cells with the retroviral supernatant (spinoculation). After reconstitution of human bone marrow implanted in SCID mice (SCID-hu bone) with the transduced HSC/progenitor cells, a significant number of donor-derived CD14+ bone marrow cells were found to express the RevM10/lyt2 gene. Finally, replication of a macrophage-tropic human immunodeficiency virus-type 1 (HIV-1) isolate was greatly inhibited in the lyt2+/CD14+ cells differentiated from transduced CD34+ cells after the enrichment of lyt2+ population. Thus, the RevM10 gene did not appear to inhibit the differentiation of HSC/progneitor cells into monocytes/macrophages. The level of retrovirus-mediated RevM10 expression in monocytes/macrophages derived from transduced HSCs is sufficient to suppress HIV-1 replication.