CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as suppressors of immune mediated reactions. The analysis of Treg function in patients with autoimmune, allergic or oncogenic diseases has emerged over the past years. In the present study we describe a CFSE based protocol to measure Treg mediated suppression of CD4(+) T cells. Measuring Treg suppressive capacity towards proliferation of anti-CD3 Ab stimulated CD4(+)CD25(-) T cells in coculture experiments by means of a CFSE based and a classical [(3)H]thymidine incorporation assay gave similar results, provided that CD4(+)CD25(+) T cells were anergic. However, when CD4(+)CD25(+) T cells proliferated upon mitogenic stimulation, data obtained by the CFSE assay allowed the detection of a significant Treg suppression whereas this was clearly underestimated using the [(3)H]thymidine assay. In addition, an indirect CFSE based method was developed to analyze antigen specific responses of total CD4(+) T cells and Treg depleted CD4(+) T cells (i.e. CD4(+)CD25(-) T cells). Our results indicate that, in healthy individuals, CD4(+) T cell responses against the multiple sclerosis (MS) auto-antigens, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), were increased in Treg depleted CD4(+) T cells as compared to total CD4(+) T cells. Our initial data suggest that Tregs in MS patients show an impaired suppression of myelin reactive T cells when compared to healthy controls. Moreover, this experimental setup permits the measurement of cytokine production of the antigen proliferated CFSE(low) T cells by additional flow cytometric analyses. In conclusion, the described CFSE based Treg suppression assay is a valuable tool to study suppressor T cells in (auto)immune disorders.