Chimerism and tolerance after bone marrow transplantation provide excellent conditions for adoptive immunotherapy with T cells of the marrow donor. We studied adoptive immunotherapy in dog leukocyte antigen-identical canine littermate chimeras. Mixed chimeras were produced by conditioning treatment with total body irradiation of a dose of 10 Gy, a uniformly lethal dose in dogs, and infusion of between 1 x 10(8) and 2 x 10(8)/kg mononuclear marrow cells treated with absorbed antithymocyte globulin for inactivation of T cells. Donors were of opposite sex. Persistent mixed chimerism was induced in six of nine dogs, chimerism was complete in one dog, and only transient in two dogs. Tolerance to donor skin grafts was demonstrated in eight dogs, including a dog without cytogenetic evidence of chimerism. Lymphocytes of the marrow donor (between 3.2 x 10(8)/kg and 4.1 x 10(8)/kg) were transfused at various times after transplantation. Nontransfused dogs survived without graft-versus-host disease (GVHD), whereas dogs transfused on days 1 and 2 and dogs transfused on days 21 and 22 developed GVHD and died. In contrast, dogs transfused on days 61 and 62 or later survived without GVHD. Chimerism converted from mixed to complete in six of six transfused dogs and in one of eight nontransfused dogs (P<0.005). Donor lymphocyte transfusions 2 years and 4.5 years after transplantation induced split chimerism with lymphoid cells of donor origin and myeloid cells of host origin in one dog and complete chimerism in the other dog. Before lymphocyte collection, donors were immunized against tetanus toxin. Seven days after lymphocyte transfusion, recipients were given booster injections of tetanus toxoid and primary immunization against diphtheria toxin. In transfused animals, antibody titers against tetanus were demonstrated already before the booster injection. Transfused animals developed higher titers of antibody against tetanus and diphtheria toxin than nontransfused animals. Donor lymphocytes converted mixed chimerism into complete chimerism without producing GVHD, when the transfusion was delayed for 2 months or later after transplantation. Transfusion of donor lymphocytes transferred immune reactivity against tetanus toxin and improved reactivity against diphtheria toxin as a new antigen.