CD4+ CD25+ T cells are critical mediators of peripheral immune tolerance. However, many developmental and functional characteristics of these cells are unknown, and knowledge of human regulatory T cells is particularly limited. To better understand how human CD4+ CD25+ T cells develop and function, we examined the diversity of CD4+ CD25+ and CD4+ CD25− T cell repertoires in both thymus and peripheral blood. Levels of T receptor excision circles (TREC) were comparable in purified CD4+ CD25+ and CD4+ CD25− thymic populations, but were significantly higher than those in samples derived from peripheral blood, consistent with murine studies demonstrating thymic development of CD4+ CD25+ regulatory T cells. Surprisingly, CD4+ CD25− T cells isolated from peripheral blood had greater TREC quantities than their CD4+ CD25+ counterparts, supporting the possibility of extrathymic expansion as well. CD4+ CD25+ and CD4+ CD25− T cells from a given individual showed overlapping profiles with respect to diversity by Vβ staining and spectratyping. Interestingly, CD4+ CD25+ T cells have lower quantities of CD3 than CD4+ CD25− T cells. Collectively, these data suggest that human CD4+ CD25+ T cells recognize a similar array of Ags as CD4+ CD25− T cells. However, reduced levels of TCR on regulatory T cells suggest different requirements for activation and may contribute to how the immune system regulates whether a particular response is suppressed or augmented.