We evaluated the efficacy of a new protocol using cyclophosphamide (CYP), granulocyte colony-stimulation factor (G-CSF) and FK506 to induce high level chimerism following rat whole-limb allotransplantation. The present study investigated the dose requirement and toxicity of CYP monotherapy in inducing stable bone marrow chimerism. Fifty-six whole-limb allotransplants from LacZ transgenic rats to LEW rats were performed. CYP at a dose of 100 mg to 200 mg/kg was injected 2 days before transplantation and G-CSF of 25 microg/kg/day was given for 4 days. FK506 was used for 28 days at 1 mg/kg/day. The level of chimerism was evaluated by semi-quantitative polymerase chain reaction. The survival of limb allografts in recipients treated with CYP of 150 mg/kg was significantly prolonged to 107 days. The onset of rejection was more prolonged to 158 days in recipients with CYP of 200 mg/kg, with two of eight grafts surviving >1 year and three recipients (38%) showed chronic, nonlethal GVHD with a high level of bone marrow chimerism. Limb allografting could contribute to chimerism in the recipient. Pretreatment with CYP had the dose-dependent effects of prolonging the survival of limb allografts. A CYP dose of 200 mg/kg appears to significantly prolong limb graft survival but frequently causes chronic nonlethal GVHD in the longer surviving recipients.