CD4+CD25+ regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred Kd-specific, CD4+ TCR (TCR75) transgenic mice to Foxp3gfp knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3gfp.TCR75 mice did not express significant numbers of natural FoxP3+ Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with Kd plus TGF-β. The resulting GFP+ TCR75 cells were anergic, whereas the GFP- TCR75 cells proliferated upon restimulation with Kd peptide. Yet both exhibited severely reduced expression of intracellular IFN-γ and TNF-α upon restimulation. GFP+, but not GFP-, TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP + TCR75 cells also inhibited polyclonal C57BL/6 anti-Kd CTL responses if the APC expressed Kd and both MHC class I and class II, and responses by OT1 T cells to B6.Kd.OVA but not B6.K d plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses. Copyright © 2007 by The American Association of Immunologists, Inc.