Several immunoregulatory mechanisms are proposed to be effective both in human and experimental Trypanosoma cruzi infection. However, the role of CD4⁺CD25^high T cells in Chagas disease has not yet been elucidated. These cells are critical for the regulation of immune response to infectious agents and in the control of autoimmune diseases. In this study, the presence of CD4⁺CD25^high regulatory T cells in the whole blood of non-infected individuals (NI), and patients with the indeterminate (IND) and cardiac form (CARD) of Chagas disease was evaluated. To further characterize this population of regulatory cells, the co-expression of CTLA-4, CD62L, CD45RO, CD45RA, HLA-DR, CD40L, CD69, CD54, IL-10R and the intracellular molecules FOXP3 and IL-10 on the CD4⁺CD25^high T lymphocytes was examined. FOXP3 was expressed by the majority of CD4⁺CD25^high when compared with the other CD4⁺ T cells subsets in patients with Chagas disease. Patients with the IND form of the disease had a higher frequency of circulating regulatory CD4⁺CD25^high T cells than patients with the CARD form. Moreover, there was an increase in CD4⁺CD25^highFOXP3+ cells that were also IL-10⁺ in the IND group whereas, in the CARD group, there was an increase in the percentage of CD4⁺CD25^high FOXP3⁺ cells that expressed CTLA-4. These data suggest that IL-10 produced by regulatory T cells is effective in controlling disease development in patients with the IND form. However, in individuals with the CARD form of the disease, the same regulatory mechanism, mediated by IL-10 and CTLA-4 expression is not sufficient to control the progression of the disease. The data suggest that CD4⁺CD25^highFOXP3⁺ regulatory T cells in patients with Chagas disease might play a role in the immune response against T. cruzi infection although with distinct effects in patients with the IND and CARD forms of disease.