CD4⁺ CD25^bright+ FoxP3⁺ regulatory T cells (Tregs) may control donor‐specific allogeneic responses in kidney transplant recipients. Recent evidence demonstrated that three phenotypical Treg‐subsets, naive (CCR7⁺CD45RO⁻), central‐memory (CCR7⁺CD45RO⁺) and effector‐memory (CCR7⁻CD45RO⁺), are essential for the development and function of antigen‐specific suppression in the lymphoid and peripheral tissues. Also, it has been appreciated that Tregs are affected by immunosuppressive agents. In clinical practice, however, the effect of a single drug remains to be determined. Therefore, we analyzed the effect of several immunosuppressive agents on the number, phenotype and function of peripheral Tregs from 46 stable kidney transplant recipients. These patients were converted to monotherapy with tacrolimus (n = 15), rapamycin (n = 17) or mycophenolate mofetil (n = 14). Blood was obtained at inclusion and 6 months thereafter. The number of Tregs increased significantly in patients on monotherapy with rapamycin (P < 0.001), which was caused by increased numbers of Tregs with a central‐memory and an effector‐memory phenotype (both P < 0.05). At 6 months after conversion, however, the suppressive function of Tregs did not significantly change in co‐cultures stimulated with donor‐Ag. Therefore, monotherapy with rapamycin allows the signals that are needed to increase the number of functional Tregs with a memory phenotype, thereby enhancing the potential capacity to regulate donor‐specific responses in the lymphoid and the peripheral tissues.