It has been known for decades that circulating human CD4 cells can express functional MHC class II molecules that induce T cell nonresponsiveness with Ag presentation. Because there is significant expression of MHC class II (MHC-II) determinants (DR) on a subpopulation CD4+CD25high regulatory T cells (Treg), we examined the function of CD4 cells expressing MHC-DR. We demonstrate that MHC-II expression on human CD4+CD25high T cells identifies a functionally distinct population of Treg that induces early contact-dependent suppression that is associated with high Foxp3 expression. In striking contrast, MHC-II− CD4+CD25high Treg induce early IL-4 and IL-10 secretion and a late Foxp3-associated contact-dependent suppression. The DR expressing CD25high Treg express higher levels of Foxp3 message and protein, compared with the DR−CD25high Treg population. Direct single-cell cloning of CD4+CD25high Treg revealed that, regardless of initial DR expression, ex vivo expression of CD25high, and not DR, predicted which clones would exhibit contact-dependent suppression, high levels of Foxp3 message, and an increased propensity to become constitutive for DR expression. Thus, the direct ex vivo expression of MHC-II in the context of CD25high identifies a mature, functionally distinct regulatory T cell population involved in contact-dependent in vitro suppression.