CD4+ CD25+ T cells have been proposed as the principal regulators of both self-tolerance and transplantation tolerance. Although CD4+ CD25+ T cells do have a suppressive role in transplantation tolerance, so do CD4+ CD25− T cells, although 10-fold less potent. Abs to CTLA-4, CD25, IL-10, and IL-4 were unable to abrogate suppression mediated by tolerant spleen cells so excluding any of these molecules as critical agents of suppression. CD4+ CD25+ T cells from naive mice can also prevent rejection despite the lack of any previous experience of donor alloantigens. However, this requires many more naive than tolerized cells to provide the same degree of suppression. This suggests that a capacity to regulate transplant rejection pre-exists in naive mice, and may be amplified in “tolerized” mice. Serial analysis of gene expression confirmed that cells sorted into CD4+ CD25+ and CD4+ CD25− populations were distinct in that they responded to TCR ligation with very different programs of gene expression. Further characterization of the differentially expressed genes may lead to the development of diagnostic tests to monitor the tolerant state.