Regulatory T cells have been reported to enhance survival of transplanted allografts. We have recently identified and cloned a novel CD3(+)CD4(-)CD8(-) (double negative, DN) regulatory T cell from mice that were given a single class I mismatched donor lymphocyte infusion and permanently accepted donor-specific skin allografts. When infused into naïve syngeneic mice, these DN T cells prolonged the survival of class I mismatched donor skin allografts. Here we further characterize the nature and mechanism of DN T-cell mediated suppression. This present study reveals that DN T cells are able to specifically eliminate activated syngeneic CD8(+) T cells that share the same T cell receptor (TCR) specificity as DN T cells in vitro. Similarly, we found that, along with an increase of recipient DN T cells in the peripheral blood, anti-donor CD8(+) T cells were also eliminated in vivo following a donor lymphocyte infusion. We further demonstrate that DN T regulatory cells do not mediate suppression by competition for growth factors or antigen presenting cells (APC) nor by modulation of APC, but require cell contact with the activated target CD8(+) T cells. This contact can be mediated either by the TCR on CD8(+) T cells that recognize constitutively expressed or acquired MHC molecules on DN T cells, or by the TCR on DN T cells that recognize constitutively expressed MHC molecules on CD8(+) T cells. Together, these data extend our previous findings, and expand the conditions in which DN T cells can potentially be used to specifically suppress allogeneic immune responses.