CD4+CD25+ regulatory T cells (Tregs) are critical for peripheral tolerance and prevention of autoimmunity. In vitro coculture studies have revealed that increased costimulation breaks Treg-mediated suppression in response to anti-CD3 or antigen. However, it was unclear whether loss of suppression arose from inactivation of Tregs or whether increased stimulation caused Th cells to escape suppression. We have investigated conditions that allow or override Treg-mediated suppression using DO11.10 TCR-transgenic T cells and chicken ovalbumin peptide 323-339-pulsed antigen-presenting cells. Treg suppression of Th proliferation is broken with potent stimulation, using activated spleen cells and high antigen dose, but is intact at low antigen dose. Costimulation with CD80 and CD86 expressed on activated dendritic cells was essential for Th cell escape from suppression at a high antigen dose. Potently stimulated Tregs were functional since they reduced levels of IL-2, IFN-gamma, IL-4 and Th CD25 expression in cocultures. Furthermore, Tregs responding to high antigen dose and activated splenocytes retained the ability to suppress proliferation, but only of Th cells responding to a sub-optimal dose of independent antigen. Together, our results demonstrate that under conditions of strong antigen-specific stimulation, Tregs remain functional, but Th cells escape Treg-mediated suppression.