Experimental animal models for autoimmunity have demonstrated the existence and crucial role of CD4⁺CD25⁺ T regulatory (Tr) cells in suppressing autoreactive T cells and promoting peripheral tolerance. Recent in vitro functional studies showed that Tr cells are enriched in the CD25^high cell population among CD4⁺ T cells, and that they totally inhibit proliferation and cytokine secretion by CD4⁺ T cells. It is not yet known if circulating Tr cells are involved in multiple sclerosis (MS). This study was done firstly to determine whether alterations of the CD4 ⁺ CD25^high T cells occur in MS, examining their frequencies. As it was reported that the suppressive activity of CD4⁺CD25⁺ Tr cells is mainly through cell surface contact pathway, we secondly analyzed the expression of the functionally important cell surface molecules of CD4⁺CD25^high Tr cells. Two- or three-colour flow cytometry was used to identify and quantify CD4⁺CD25⁺ Tr cells and CD4⁺CD25^high Tr cells among blood CD4⁺ T cells in MS patients without treatment vs. patients treated with either interferon-β (IFN-β) or glatiramer acetate (GA) or IFN-β + GA in combination vs. healthy controls (HC). Expression of functionally important surface molecules CD45RO, CD69, CD95, HLA-DR, and intracellular CTLA-4 and IL-10 production by CD4⁺CD25^high Tr cells were investigated. CD4⁺CD25⁺ T cells constituted around 6% of CD4⁺T cells in all MS patient groups, and 7% in HC. There were also no changes in the proportions of CD4⁺CD25⁺ Tr cells and CD4⁺CD25^high Tr cells in a longitudinal follow-up of MS patients before and during IFN-β treatment. Frequencies of circulating CD4⁺CD25^highTr cells among CD4⁺ T cells were also similar and their surface or intracellular molecular expression did not vary in MS patients, irrespective of treatment, compared to HC. This study suggests that levels of circulating CD4⁺CD25⁺ Tr cells and CD4⁺CD25^high Tr cells are not altered in MS, and are unaffected by substances currently used to modulate the disease.