Naturally occurring regulatory T cells (Tregs), residing in CD4+CD25+ fraction, are important in the maintenance of immune homeostasis. One of the functional characteristics of Tregs is close relationship between suppressive activity and anergy in vitro. Meanwhile, many in vitro assays have observed Treg proliferation and suppressive activities in different settings, i.e., in the absence and presence of CD25(-) responder cells. If the presence of responder cells affect the proliferation of Tregs, comparison between the two settings would be inappropriate. In the present study, we traced proliferation as well as suppressive activities of Tregs in the same setting of coculture in response to varying concentrations of anti-CD3 and anti-CD28. Quantitative analysis using two parameters, precursor frequency and CD25 mean fluorescence intensity, reflecting early and late proliferative responsiveness, respectively, showed that proliferation of Tregs was dependent on the responder cells and proliferating Tregs preserved suppressive activities. Transwell assay and neutralization assay showed that the enhancement of Treg proliferation by the responder cells was mediated through secreted IL-2. Quantitative analysis also showed distinct mode of suppression by Tregs according to the presence or absence of anti-CD28. In the absence of anti-CD28, Tregs suppressed the initial proliferation, whereas in the presence of anti-CD28, Tregs suppressed only the late expansion of the responder cells by lowering CD25 expression. Considering that Tregs cannot produce IL-2 by themselves while they constitutively express CD25 (IL-2Ralpha), dependency of Tregs on their target of suppression (responder cells) for proliferation supports the model for feedback loop of immune regulation by Tregs.