CD25⁺ regulatory T cells (Treg) are a heterogeneous population that exists as CD44^low and CD44^high cells. Here we report that while both CD44^low and CD44^high Treg are anergic and express similar levels of Foxp3, CD44^high Treg are highly proliferative in vivo and are more potent suppressors in vitro than CD44^low Treg. From analysis of the properties of Treg derived from germ‐free mice, it was concluded that peptide antigens derived from intestinal microorganisms are not essential for the generation, in vivo proliferation or suppressive activity of Treg. Our results suggest that gut flora antigens play little or no role in the heterogeneity and homeostatic regulation of Treg.