The mechanism and stimulatory requirements of regulatory T cell (Treg)-mediated suppression are still unclear. To assess the requirement for Treg stimulation by cognate peptide:MHC, we used T cells from OTII and AND TCR transgenic mice that are specific for and restricted by distinct, noncrossreactive peptide:MHC combinations. This allowed us to independently activate Tregs and their conventional T cell (Tconv) targets. Surprisingly, we found that suppression can occur in the absence of peptide:MHC-mediated stimulation of Tregs. This suppression was Treg dependent and not due to cold target inhibition. Using Rag1(-/-) TCR transgenic T cells, we show that regulation of Tconv proliferation by heterogeneous Tregs is not due to alloreactivity or crossreactivity. Finally, using anti-TCR-Vbeta8-coated microbeads and Vbeta8(-) Tregs, we show that TCR stimulation-independent suppression can occur in the absence of APCs. These data suggest that Tregs may possess constitutive regulatory activity that can be mediated in the absence of cognate peptide:MHC-TCR stimulation.