SUMMARYPrevention of canine graft-versus-host disease (GVHD) was attempted with in vitro treatment of the marrow graft with absorbed rabbit anti-dog thymocyte globulin (aATG). Cross-reactive antibodies against hemopoietic stem cells were eliminated following absorption with liver and kidney homogenate and spleen cells of newborn dogs. The growth of granulocytic colonies was almost completely inhibited by prior incubation of marrow with crude antithymocyte globulin (ATG) in the presence of complement, but not by incubation with aATG. In vitro treatment of marrow with aATG did not interfere with its ability to reconstitute a lethally irradiated autologous host. Allogeneic transplantations were performed in lethally irradiated DLA heterozygous recipients with marrow of compatible, DLA homozygous littermates.Transplantation of untreated marrow resulted in death from GVHD of five of five recipients. In vitro treatment of marrow with aATG at a concentration of 1:100 (v/v) prevented GVHD and induced stable graft-versus-host tolerance in one dog, delayed GVHD in two dogs, and interfered with hemopoietic engraftment in two dogs. Treatment with aATG at a concentration of 1:200 induced tolerance in three dogs and prolonged survival in two. The four surviving chimeras are alive 240 to 380 days after transplantation without ever having shown symptoms of GVHD. The potential value of in vitro treatment of marrow with aATG in clinical bone marrow transplantation is discussed.