It has been demonstrated that T cells with regulatory properties are present within the peripheral blood CD4⁺CD25⁺ T cell compartment. Here, we describe an original method to purify human CD4⁺CD25⁺CD152⁺ T lymphocytes as living cells by forcing the exportation of CTLA‐4 molecules stored in intracellular vesicules at the cell surface. By doing so, we demonstrate that CD4⁺CD25⁺ T cells contain a smaller and more homogeneous population enriched in cells with in vitro regulatory activity. Moreover, we show that this enrichment in regulatory T cells is associated with an increased expression of Foxp3 and that CD4⁺CD25⁺CD152⁺ T lymphocytes display a much stronger suppressive activity in controlling in vitro proliferation of alloantigen‐specific T cells than CD4⁺CD25⁺CD152^– T lymphocytes purified in parallel. Lastly, by purifying such cells expressing CTLA‐4, we demonstrate that indeed CTLA‐4 is involved in CD4⁺CD25⁺CD152⁺ T cell regulatory activity, while suppressive cytokines are not.