Recent studies have opened the possibility that quiescent, G₀/G₁ hematopoietic stem cells (HSC) can be gene transduced; lentiviruses (such as HIV type 1, HIV) encode proteins that permit transport of the viral genome into the nucleus of nondividing cells. We and others have recently demonstrated efficient transduction by using an HIV-1-based vector gene delivery system into various human cell types including human CD34⁺ cells or terminally differentiated neurons. Here we compare the transduction efficiency of two vectors, HIV-based and murine leukemia virus (MuLV)-based vectors, on untreated and highly purified human HSC subsets that are virtually all in G₀/G₁. The HIV vector, but not MuLV vector supernatants, transduced freshly isolated G₀/G₁ HSC from mobilized peripheral blood. Single-step transduction using replication-defective HIV resulted in HSC that expressed the green fluorescent protein (GFP) transgene while retaining their stem cell phenotype; clonal outgrowths of these GFP⁺ HSC on bone marrow stromal cells fully retained GFP expression for at least 5 weeks. MuLV-based vectors did not transduce resting HSC, as measured by transgene expression, but did so readily when the HSC were actively cycling after culture in vitro for 3 days in a cytokine cocktail. These results suggest that resting HSC may be transduced by lentiviral-based, but not MuLV, vectors and maintain their primitive phenotype, pluripotentiality, and at least in vitro, transgene expression.