Transplantation tolerance can be induced in the adult mouse by the selective manipulation of the CD4+ T cell subset. C3H/He recipients with prolonged survival (> 120 days) of C57BL/10 cardiac allografts induced by treatment, at the time of transplantation, with the anti-CD4 mAb, YTS 191.1, were skin grafted simultaneously with donor-specific and third-party (BALB/c) skin. The development of donor-specific tolerance was proved by the specific prolongation of C57BL/10 skin graft survival, while third-party grafts were rejected. Further investigation of recipients with long-term surviving primary heart allografts showed that donor-specific tolerance was associated with organ-specific differences. Secondary cardiac allografts were universally accepted, even at 42 days after the primary heart transplant, while prolonged survival of donor-specific skin grafts was not obtained until more than 120 days after primary cardiac transplantation. Analysis of leukocyte reactivity in the mixed leukocyte culture (MLC) showed no correlation between the proliferative response of recipient T cells in vitro to either donor or third-party alloantigen and the survival of either heart of skin allografts. These results illustrate the significant challenge presented when attempting to define and assess accurately the state of transplantation tolerance.