Repeated exposures to both microbial and innocuous Ags in vivo have been reported to both eliminate and tolerize T cells after their initial activation and expansion. The remaining tolerant T cells have been shown to suppress the response of naive T cells in vitro. This feature is reminiscent of natural CD4+ CD25+ regulatory T cells. However, it is not known whether the regulatory function of in vivo-tolerized T cells is similar to the function of natural CD4+ CD25+ regulatory T cells. In this study, we demonstrate that CD4+ CD25+ as well as CD4+ CD25− T cells isolated from mice treated with superantigen three consecutive times to induce tolerance were functionally comparable to natural CD4+ CD25+ regulatory T cells, albeit more potent. The different subpopulations of in vivo-tolerized CD4+ T cells efficiently down-modulated costimulatory molecules on dendritic cells, and their suppressive functions were strictly cell contact dependent. Importantly, we demonstrate that conventional CD4+ CD25− T cells could also be induced to acquire regulatory functions by the same regimen in the absence of natural regulatory T cells in vivo, but that such regulatory cells were functionally different.