Background. Tolerance to cardiac allografts can be induced in mice and rats by the injection of donor alloantigen into the thymus in combination with a CD4 T-cell-depleting antibody. CD8+ cells in these animals are hyporesponsive to graft-specific alloantigens. Most of the CD8+ T cells in the transgenic 2C mouse express a T-cell receptor specific for the class I major histocompatibility complex L(d+) locus. This study was designed to determine whetherthe adoptive transfer of these 2C T cells could precipitate rejection of a tolerant, completely major histocompatibility complex-mismatched L(d+) or L(d-) heart. Methods. C57BL/6 mice (L(d-)) were given 10 x 106 cells of BALB/c (L(d+)) or dm2 (BALB/c background laching L(d) [L(d-)]) splenocytes intrathymically and GK1.5 (10 mg/kg) intraperitoneally. Twenty-one days later, BALB/c or dm2 hearts were transplanted. On the day of transplantation or after long-term allograft acceptance, recipients received naive 2C cells or 2C cells sensitized by in vitro mixed lymphocyte culture with BALB/c (L(d+)). Results. Mean survival time of BALB/c cardiac allografts in untreated C57BL/6 mice was 7.3 days, although 73% of the mice that were pretreated with BALB/c splenocytes IT plus GK1.5 accepted the donor antigen-specific heart allografts indefinitely. All recipients that were pretreated with the intrathymic plus GK1.5 and that were injected with naive 2C cells at the time of heart transplantation experienced rejection of the BALB/c (L(d+)), but not the dm2 (L(d-)) hearts. In contrast, naive 2C cells could not reject tolerant (>30 days acceptance) BALB/c (L(d+)) hearts. 2C cells sensitized in vitro against L(d) were able to reject established BALB/c hearts but could not reject the L(d-) dm2 hearts. Conclusions. L(d)-specific 2C T-cell receptor transgenic T cells that are adoptively transferred to recipients will precipitate the rejection of accepted hearts that express class I L(d+) in mice rendered tolerant by an intrathymic injection of alloantigen plus anti-CD4 monoclonal antibodies.