Allograft rejection is dependent on T cell activation, which requires both the engagement of the T cell receptor by antigen in the context of the MHC molecules and costimulatory signals delivered by cell surface molecules such as B7-CD28/CTLA4 pathway. CTLA4-Ig is a fusion protein that blocks this pathway and has previously been shown to prolong both allograft and xenograft survival. The current study demonstrates markedly prolonged murine cardiac allograft survival and specific prolongation of secondary skin grafts using a combination of CTLA4-Ig plus donor bone marrow. A role for hematopoietic chimerism in the establishment of CTLA4-Ig-induced transplantation tolerance was investigated using reverse transcriptase polymerase chain reaction analysis of recipient tissues. Expression of donor-specific MHC class II transcripts in both peripheral and lymphoid tissues was demonstrated at greater than 200 days after transplant. To investigate the functional significance of this observation, heart donors, and donor bone marrow were irradiated before transplantation in CTLA4-Ig-treated recipients. A reduction in allograft survival was associated with irradiation of both the donor heart and the bone marrow. These results suggest that there may be a donor-derived radiosensitive element that enhances allograft survival in this model. Reverse transcriptase polymerase chain reaction analysis of allografts of tolerant and control animals at days 5, 8, and 12 after transplantation failed to demonstrate a dramatic difference in the expression of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma message. Cytotoxicity effector transcripts were largely intact in CTLA4-Ig + bone marrow-treated recipients as they showed no decrease in intragraft granzyme, perforin, Fas, or Fas ligand transcripts during thr first 8 days after transplant. These results imply that complex mechanisms may be important for the induction and maintenance of transplantation tolerance in the CTLA4-Ig plus bone marrow murine cardiac allograft model.