Little is known regarding the participation of CD4+ CD25+ regulatory T cells (Treg) in TCD8+ responses. In this study, we show that Treg depletion via treatment with anti-CD25 mAb (PC61) significantly enhances TCD8+ responses to influenza A virus, vaccinia virus, and SV40-transformed cells induced by either direct priming or cross-priming. PC61 did not enhance TCD8+ responses in CD4-deficient mice, providing the initial demonstration that PC61 acts on a subset of TCD4+, and not on other cells that express either CD25 or a fortuitously cross-reactive Ag. We further show that Treg selectively suppress responses to the most immunodominant TCD8+ determinants in the three systems examined. Therefore, Treg influence TCD8 immunodominance hierarchies by moderating disparities in responses to different determinants.