Abstract Introduction of MHC class I K b cDNA via recombinant retrovirus into B10.AKM (K k) bone marrow cells (BMC) has been shown to confer specific hyporesponsiveness to B10.MBR (K b) allogeneic skin grafts in lethally irradiated B10.AKM recipients of the transduced syngeneic BMC. We have recently developed a nonmyeloablative conditioning regimen that allows engraftment of fully MHC-mismatched allogeneic bone marrow and the induction of donor-specific tolerance. We ultimately plan to adapt this nonmyeloablative regimen for the use of retroviral transfer of the K b gene to syngeneic marrow. As a step toward this goal, we have assessed the effects of our current BMC transduction protocol on engraftment of class I mismatched marrow in mice prepared using the nonmyeloablative regimen. BMC from B10.MBR (K bI kD q) mice treated 2 days earlier with 5-fluorouracil (5-FU) were cultured for 4 days with rIL-3 and rIL-6, and then injected into B10.AKM (K kI kD q) recipients conditioned with anti-CD4 and anti-CD8 mAbs, 7 Gy of thymic irradiation and 3 Gy of whole body irradiation. Engraftment was comparable to that of freshly prepared normal B10.MBR marrow. All recipients of 10 6 precultured BMC developed long-term multilineage mixed WBC (white blood cells) chimerism, and six of seven of these animals showed long-term specific tolerance to B10.MBR skin grafts. Four of seven recipients of 2 × 10 5 precultured BMC showed long-term repopulation by the donor of > 1% multiple WBC lineages and four of five recipients showed specific tolerance to B10.MBR tail skin. These data suggest that our previously described nonmyeloablative conditioning regimen could be applicable to the gene therapy approach for the induction of donor-specific transplantation tolerance.