The hematopoietic stem cell has long been considered an ideal target for the introduction of therapeutic genes to treat human disorders such as Fanconi anemia (FA). Although recent progress in large animal models is encouraging, application to nonmalignant conditions is limited by the perceived necessity of myeloablative conditioning. We and others have shown that very low irradiation doses are sufficient to allow significant hematopoietic engraftment in murine hosts even after the introduction of xenogeneic genes. To determine the degree of engraftment of genetically modified cells attainable with very low irradiation doses in larger animals, we employed the rhesus macaque competitive repopulation model. Four animals underwent mobilization with stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) followed by apheresis. The apheresis product was enriched for the CD34-positive fraction by immunomagnetic selection and split equally for transduction with either G1FC26, a retroviral vector carrying the Fanconi anemia complementation group C gene, or PLII, a nonexpression control retroviral vector carrying both neomycin and beta-galactosidase gene sequences modified to prevent translation. Transductions were performed daily in the presence of fresh IL-3, IL-6, SCF, and Flt-3 ligand on fibronectin-coated plates over 96 h. Animals were conditioned with a single dose of either 100 (n = 2) or 200 (n = 2) cGy and received the combined products of transduction on the following day. None of the animals experienced clinically significant neutropenia nor required the use of central line placement, transfusional support with blood products, or intravenous antibiotics. Using real-time PCR, circulating levels of genetically modified cells as high as 1% were initially detected. Stable, albeit, significantly lower levels from both vector-transduced aliquots (<0.1%) persisted beyond 12 months posttransplant in all four animals. Although not sufficient to correct the phenotype in many human disorders, stable low-level engraftment by genetically modified cells following low-intensity conditioning may prove adequate in disorders such as FA due to the selective advantage conferred upon corrected cells.