Control of cardiac transplant rejection without toxic immunosuppressive drugs remains an unreached goal. Our laboratory and others have shown that intrathymic inoculation of donor-specific allogeneic spleen cells can produce tolerance to a subsequent cardiac allograft. The present experiments were designed to investigate whether the degree of donor-recipient histoincompatibility influenced the efficacy of this technique. Four congeneic strains of rats with different degrees of histoincompatibility were studied. Heterotopic cardiac transplantation was done with the following congeneic strain combinations: DA donor into PVG recipient (full major histocompatibility complex and nonmajor histocompatibility complex incompatibility); PVG.RT1a donor into PVG recipient (full major histocompatibility complex incompatibility); PVG.RT1a donor into PVG.R1 recipient (partial major histocompatibility complex incompatibility). Prospective graft recipients underwent intraperitoneal injection of 1 ml antilymphocyte serum and intrathymic injection of 5 x 10(7) prospective donor spleen cells. Three weeks later, heterotopic cardiac transplantation was done with a heart from a donor of the same strain as that used to obtain splenocytes for intrathymic injection. Prolongation of graft survival was observed in pretreated recipients in all strain combinations but was greatest in recipients that differed from donors at fewer histocompatibility loci. Complete graft tolerance was not seen in strain combinations that included nonmajor histocompatibility complex incompatibilities. DA heart survival in PVG recipients was 50.6 days (p < 0.04 versus controls); PVG.RT1a graft survival in PVG hosts was 165.8 days (p < 0.02 versus control) and in PVG.R1 recipients 163.8 days (p < 0.02 versus controls) with four of five grafts in each group surviving indefinitely (more than 200 days).