Recent insight into the balance of self-tolerance and auto-aggression has raised interest in using human regulatory T (Treg) cells for adoptive immunotherapy of unlimited autoimmune diseases including type-1 diabetes, rhematoid arthritis and multiple sclerosis. The therapeutic use of Treg cells, however, is so far hampered by the inefficiency of current protocols in making them accessible for genetic manipulations. We report here that TCR/CD3 stimulation that is accompanied by extensive CD28 costimulation makes human Treg cells susceptible to retroviral gene transfer ex vivo while preserving their properties in vitro and in vivo. To show the power of genetic manipulation of human Treg cells, we engineered ‘designer Treg cells’ by retroviral expression of a chimeric immunoreceptor with defined specificity, which activates Treg cells in a ligand-dependent manner to proliferate, to secrete high amounts of interleukin-10 and to repress an ongoing cytolytic T-cell response in vivo. The procedure in genetically modifying human Treg cells ex vivo will open a panel of applications for their use in the adoptive therapy of deregulated immune responses.