Immunosuppressive drugs are essential for the prevention of acute transplant rejection but some may not promote long-term tolerance. Tolerance is dependent on the presence and regulatory function of CD4+CD25+ T cells in a number of animal models. The direct effects of immunosuppressive drugs on CD4+CD25+ cells, particularly those that interfere with IL-2 signaling are uncertain. We studied the effects of the rapamycin derivative everolimus and the anti-CD25 monoclonal antibody basiliximab on the regulatory capacity of human CD4+CD25+ cells in vitro. Both drugs permitted the suppression of proliferation and IFN-γ secretion by CD4+CD25− cells responding to allogeneic and other polyclonal stimuli; CTLA-4 expression was abolished on CD4+CD25+ cells without compromising their suppressive ability. Everolimus reduced IFN-γ secretion by CD4+CD25− cells before the anti-proliferative effect: this is a novel finding. Exogenous IL-2 and IL-15 could prevent the suppression of proliferation by CD4+CD25+ cells and the drugs could not restore suppression. By contrast, suppression of IFN-γ secretion was only slightly impeded with the exogenous cytokines. Finally, CD4+CD25+ cells were more resistant than CD4+CD25− cells to the pro-apoptotic action of the drugs. Together these data suggest that CD4+CD25+ cells may still exert their effects in transplant patients taking immunosuppression that interferes with IL-2 signaling.