Gene replacement therapy for diseases of the hematopoietic system requires efficient gene transfer to pluripotent hematopoietic stem cells. We have systematically compared a number of protocols for retrovirus-mediated gene transfer into murine repopulating hematopoietic stem cells. Recipients of infected bone marrow cells were analyzed for the presence of the transduced provirus 4 months after transplantation. Our results show that 5-fluorouracil (5-FU) pretreatment of donor animals was required for efficient gene transfer and that 5-FU-treated bone marrow retained more repopulating activity in culture than untreated bone marrow. A comparison of retrovirus-mediated gene transfer by co-cultivation of bone marrow cells with retrovirus producer cells as opposed to gene transfer by culturing bone marrow cells in retrovirus-containing supernatant revealed that gene transfer by cocultivation was more efficient than supernatant infection. However, the repopulating ability of bone marrow cells cocultured with retrovirus producer cells was reduced compared to bone marrow cells cultured in virus-containing medium.