We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringent in vitro assays of primitive, quiescent human hematopoietic progenitor cells. Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34⁺ cells (45.5% GFP⁺) and in CD34⁺CD38⁻ cells in G₀ (12.4% GFP⁺), 48 hr after transduction. However, GFP could also be detected short-term in CD34⁺ cells transduced with a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34⁺ cells. The MLV vector did not transduce more primitive, quiescent CD34⁺CD38⁻ cells (n = 8). In contrast, stable transduction of CD34⁺CD38⁻ cells by the lentiviral vector was seen for over 15 weeks of extended long-term culture (9.2 ± 5.2%, n = 7). GFP expression in clones from single CD34⁺CD38⁻ cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce CD34⁺ and CD34⁺CD38⁻ cells (13.5 ± 2.5%, n = 11 and 12.2 ± 9.7%, n = 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.