CD4⁺CD25⁺ T cells play a central role in the suppression of autoimmunity and inflammation, making their in vivo expansion a highly attractive therapeutic target. By phenotyping with a novel rat CTL antigen‐4 (CTLA‐4)‐specific monoclonal antibody (mAb) and functional in vitro assays, we here first establish that rat CD4⁺CD25⁺ T cells correspond to the regulatory T cells (Treg cells) described in mice and humans: they constitutively express CTLA‐4, produce IL‐10 but not IL‐2, and are able to suppress the proliferation of costimulated CD25‐negative indicator cells. Furthermore, we show that rat Treg cells respond less well than CD25^– T cells to conventional costimulation, but are readily expanded in vitro with "superagonistic" CD28‐specific mAb which are potent mitogens for all T cells without the need for TCR engagement. In vivo, functional Treg cells are preferentially expanded by CD28 stimulation over other T cell subsets, leading to a 20‐fold increase within 3 days in response to a single antibody dose. These data suggest that CD28‐driven activation of Treg cells may be highly effective in the treatment of inflammatory and autoimmune diseases.