Previously, we have demonstrated that pretransplant donor lymphocyte infusion (DLI) can activate recipient-derived CD3+CD4-CD8- double negative T regulatory (DN Tr) cells which have a potent immune regulatory function in vitro and in vivo. Here we studied the regulatory ability of DN T cell clones generated from the spleens of nai;ve anti-L(d) transgenic TCR+ (2C x dm2)F1 mice. We were able to identify subsets of DN T cell clones that were able to kill anti-Ld CD8+ T cells, and therefore had regulatory properties, and DN T cells with no regulatory properties. Next, we investigated the ability of these in vitro generated DN T cell clones to enhance cardiac allograft survival. (2C x dm2)F1 transgenic mice were infused with either regulatory or non-regulatory DN T cell clones, or left untreated one day before receiving an Ld-mismatched cardiac grafts from (C57BL/6 x Balb/c)F1 mice. Injection of non-regulatory DN T clone cells did not prolong cardiac graft survival in (2C x dm2)F1 mice when compare to untreated controls. In contrast, all of the cardiac grafts survived more than 100 days in mice that received DN Tr clone cells prior to transplantation. These results demonstrate that DN Tr cells can be generated in vitro and protect cardiac allograft from rejection when infused into recipients prior to transplantation. They also suggest that DN Tr cells may provide a novel therapy for the treatment of allograft rejection.