Regulatory T cells (Tregs) limit autoimmunity but also attenuate the magnitude of antipathogen and antitumor immunity. Understanding the mechanism of Treg function and therapeutic manipulation of Tregs in vivo requires identification of Treg-selective receptors. A comparative analysis of gene expression arrays from antigen-specific CD4(+) T cells differentiating to either an effector/memory or a regulatory phenotype revealed Treg-selective expression of LAG-3, a CD4-related molecule that binds MHC class II. Antibodies to LAG-3 inhibit suppression by induced Tregs both in vitro and in vivo. Natural CD4(+)CD25(+) Tregs express LAG-3 upon activation, which is significantly enhanced in the presence of effector cells, whereas CD4(+)CD25(+) Tregs from LAG-3(-/-) mice exhibit reduced regulatory activity. Lastly, ectopic expression of LAG-3 on CD4(+) T cells significantly reduces their proliferative capacity and confers on them suppressor activity toward effector T cells. We propose that LAG-3 marks regulatory T cell populations and contributes to their suppressor activity.