Background.We have recently established a new method for bone marrow transplantation (BMT) in mice: bone marrow cells are directly injected into the intra-bone marrow (IBM) cavity. IBM-BMT induces persistent donor-specific tolerance and enhances the rapid recovery or reconstitution of the hematolymphoid system of donor origin without any signs of graft-versus-host disease (GVHD) or graft failure. Furthermore, the prior injection of fludarabine can reduce the irradiation dose to the sublethal level (4.5 Gy×2). Therefore, we hypothesize that IBM-BMT plus fludarabine is applicable to allogeneic leg transplantation in rats.Methods.Brown Norway (BN; RT1An) rats were injected intravenously with 50 mg/kg of fludarabine phosphate, followed by sublethal fractionated irradiation (4.5 Gy×2) 1 day before IBM-BMT. The hind limbs from Fischer 344 (F344; RT1Al) rats were transplanted on day 0, and bone marrow cells (3×107cells/50 &mgr;L) obtained from the donor F344 rats were injected into the bone marrow cavity of the left tibias of the recipient BN rats.Results.The hematolymphoid cells in the recipient BN rats were completely reconstituted by the cells of the donor F344 rats. The limbs transplanted from the donor F344 rats were accepted for >1 year without any clinical signs of rejection (10 of 10). The lymphocytes of the BN rats showed tolerance to both donor-type and recipient-type major histocompatibility complex determinants in mixed lymphocyte reaction, but showed a significant response to the third-party major histocompatibility complex determinants.Conclusions.Using a combination of the injection of fludarabine, low-dose irradiation, and IBM-BMT, we have succeeded in allogeneic limb transplantation without using any immunosuppressants after the operation. This strategy would be applicable to the transplantation of other vascularized organs in humans.