SUMMARY Previous studies in dogs have shown that a single preceding transfusion with whole blood from the intended marrow donor can immunize a recipient and lead to rejection of a subsequent marrow graft even when donor and recipient are littermates compatible for the major dog leukocyte antigen (DL-A) locus. The present study investigated two approaches to diminish the chance of marrow graft rejection in transfused canine recipients: (1) the use of the immunosuppressive drug methotrexate (MTX) after grafting, and (2) the use of buffy coat-poor blood instead of whole blood for the preceding transfusion. Recipients were conditioned for marrow grafting by 1,200 R of total body irradiation followed within 4 hr by infusion of hemopoietic cells from DL-A-incompatible unrelated donors. MTX, 0.4–0.5 mg/kg, was administered i.v. on days 1, 3, 6, and 11 after irradiation and grafting and then once weekly until day 102. A single preceding transfusion of whole blood from the intended DL-A-incompatible marrow donor in most instances led to rejection of the marrow graft. Treatment with MTX did not diminish the incidence of graft rejection. Substituting buffy coat-poor blood (reduction in white blood cells: 99.8 ± 0.2% (SD) and platelets: 99.1 ± 0.7% (SD)) for whole blood did not significantly change the incidence of immunization of recipients.