To track the changes in the tested Treg markers especially Foxp3 following activation to determine whether data of human studies using Foxp3 in evaluation of Tregs are reliable or not. Four-colour flow cytometry analysis was carried out to calculate the percentages of Tregs before and after lymphocyte activation. Foxp3 expression by CD4(+)CD25(+)* and CD4(+)CD25(high) T cells increased after T cell activation. A moderate negative correlation was observed between the percentage of each of CD4(+)CD25(+)Foxp3(+)IL10(+) or CD4(+)CD25(high) Foxp3(+)IL10(+) T cells and the percentage of CD4(+)CD25(+) T cells "after activation" and a weak negative correlation was similarly observed between the percentage of CD4(+)CD25(-)Foxp3(+)IL10(+) T cells and the percentage of CD4(+)CD25(+) T cells "after activation". A moderate negative correlation was observed between the percentage of each of CD4(+)CD25(+)Foxp3(+)IL10(+), CD4(+)CD25(high)Foxp3(+)IL10(+) or CD4(+)CD25(-) Foxp3(+)lL10(+) T cells and the percentage of CD4(+)CD25(high) T cells "after activation". CD4(+)CD25(high) T cell subpopulation expressed a significantly higher level of intracellular Foxp3 compared with CD4(+)CD25(low) and CD4(+)CD25(-) T cells subpopulations. In conclusions, Foxp3 is a good marker of Tregs especially if panels of markers were used for their identification. CD4(+)CD25(high) Foxp3(+) T cell subpopulation mostly represents Tregs and thus should be the one targeted in Treg studies.