Histoincompatibility between murine donors and recipients of bone marrow (BM) transplants reduces engraftment, and this compromises assessment of hematopoietic stem cells (HSCs) in certain transgenic mice. To study HSCs in the S+S-Antilles mouse model of human sickle cell disease (SCD), we developed an autotransplant protocol. Initial experiments showed no differences between S+S-Antilles mice and normal C57BL/6 (+/+) mice in their radiosensitivity or baseline hematopoietic progenitor numbers. The kinetics of red blood cell (RBC) replacement post-transplant in +/+ recipients of mixtures of transgenic and +/+ BM cells also showed no competitive advantage of the +/+ cells. BM cells were then aspirated from mice 4 days after 5-fluorouracil treatment, transduced with a green fluorescent protein (GFP)-encoding retrovirus, and transplanted into the same recipients that, just before transplant, were irradiated with 800 cGy. We subsequently detected high levels of GFP(+) RBCs (21-79%) and white blood cells (WBCs; 35-88%) in the blood for 11 months and showed that transduced HSCs regenerated in the primary mice also repopulated secondary mice. These findings provide a generally applicable protocol for performing autotransplants in mice and forecast the potential utility of this approach in assessing HSC-based gene therapy protocols in transgenic mouse models of many human diseases.