Although CD4⁺CD25⁺ regulatory T cells are pivotal in the prevention of autoimmunity and appear to mediate transplantation tolerance, little is known concerning their antigen specificity. Here we describe the induction of a human CD4⁺CD25⁺ regulatory T-cell line specific for a defined peptide alloantigen (human leukocyte antigen A2 [HLA-A2] 138-170) by priming purified CD4⁺CD25⁺ cells ex vivo. The regulatory cells were anergic and retained their ability to suppress antigen-driven responses of CD4⁺CD25^– cells. They inhibited not only interleukin 2 (IL-2) secretion by CD4⁺CD25^– T cells specific for the same peptide but also direct alloresponse of naive CD4⁺CD25^– T cells stimulated by semiallogeneic dendritic cells (DCs) in the presence of the peptide (“linked suppression”). They also suppressed the response of CD4⁺ T cells specific for viral and bacterial antigens. The suppressive T-cell line showed sustained high CD25 expression. These findings suggest that peripheral CD4⁺CD25⁺ regulatory cells are a precommitted cell lineage from which cells with specificity for non–self-peptides can be selected. This may pave the way for inducing and expanding peptide antigen-specific regulatory T cells ex vivo for cell therapy in transplantation, allergy, and autoimmune disease.