We examined the effect of methotrexate (MTX) and cyclosporin A (Cy A) on engraftment, graft-versus-host disease (GVHD), and the induction of tolerance in dogs prepared for marrow transplantation by 9 Gy of total body irradiation and grafted with bone marrow and buffy coat cells. Nineteen dogs were given grafts from DLA-identical littermates followed by immunosuppression with Cy A for 25 or 100 days. All had sustained engraftment, and 12 became healthy long-term chimeras. Sixty dogs were given grafts from DLA-nonidentical unrelated donors. Among nine given MTX only postgrafting, one rejected the graft nd eight died with GVHD. Among 18 dogs given Cy A only postgrafting, eight failed to achieve engraftment, seven died of various causes, and three died with GVHD. Thirty-four dogs were given both MTX and Cy A in various regimens postgrafting. The only long-term survivors were 4 of 10 dogs given MTX on days 1, 3, 6, and 11 and Cy A from days 0 through 100. Two have chronic GVHD. We conclude that Cy A can induce graft-host tolerance across minor, but not major, histocompatibility differences. The combination of MTX early after transplantation with Cy A prevents failure of engraftment of histoincompatible marrow and some recipients become long-term survivors.