Postgrafting cyclosporine (CSP) given for 35 days resulted in establishment of stable marrow grafts from DLA-identical canine littermates after otherwise suboptimal, but nevertheless, lethal conditioning with 450 cGy of total body irradiation (TBI). We now asked whether sustained allografts could be achieved after sublethal TBI or without TBI. Five groups of recipients were studied. Dogs in group 1 were given 200 cGy TBI and postgrafting CSP, 15 mg/kg twice daily by mouth on days -1 to 35 posttransplant. Dogs in group 2 were given 200 cGy TBI and methotrexate (MTX), 0.4 mg/kg intravenously (I.V.) on days 1, 3, 6, and 11 along with CSP. Dogs in group 3 were given 200 cGy TBI and CSP along with mycophenolate mofetil (MMF), 10 mg/kg twice daily subcutaneously (S.C.) on days 0 to 27 after transplant, a novel immunosuppressive combination. Dogs in group 4 were given 100 cGy TBI and MMF/CSP. Dogs in group 5 were not given TBI and they received MMF/CSP posttransplant. Allografts were assessed by (Ca)n dinucleotide repeat polymorphism studies in cells from peripheral blood, lymph nodes, and marrow. Dogs in group 1 had transient mixed donor-host hematopoietic chimerism for no more than 4 weeks. Three of six dogs in group 2 had transient mixed chimerism for 3 to 11 weeks, and three have remained stable mixed chimeras for up to 60 weeks now. Four of five dogs in group 3 have remained stable mixed chimeras for 54 to 57 weeks now, while one lost the allograft after 12 weeks. All dogs in groups 4 and 5 rejected their allografts after 2 to 12 weeks. In summary, the establishment of stable mixed hematopoietic chimerism following nonmyelosuppressive and nontoxic conditioning programs has remained a difficult goal. Here we present evidence in a large random-bred animal species that this goal may be achievable with pharmacological immunosuppression postgrafting, capable of inhibiting both host-versus-graft (HVG) and graft-versus-host (GVH) reactions in the setting of DLA-identical grafts.