CD4⁺CD25⁺ regulatory T cells contribute to the maintenance of peripheral tolerance by active suppression because their deletion causes spontaneous autoimmune diseases in mice. Human CD4⁺ regulatory T cells expressing high levels of CD25 are suppressive in vitro and mimic the activity of murine CD4⁺CD25⁺ regulatory T cells. Multiple sclerosis (MS) is an inflammatory disease thought to be mediated by T cells recognizing myelin protein peptides. We hypothesized that altered functions of CD4⁺CD25^hi regulatory T cells play a role in the breakdown of immunologic self-tolerance in patients with MS. Here, we report a significant decrease in the effector function of CD4⁺CD25^hi regulatory T cells from peripheral blood of patients with MS as compared with healthy donors. Differences were also apparent in single cell cloning experiments in which the cloning frequency of CD4⁺CD25^hi T cells was significantly reduced in patients as compared with normal controls. These data are the first to demonstrate alterations of CD4⁺CD25^hi regulatory T cell function in patients with MS.