Gammadelta T cells play an important role in innate immunity against infections; however, the regulation of these cells remains largely unknown. In the present study, we show that ESAT-6, an antigen of Mycobacterium tuberculosis, induces IFN-gamma secretion by human gammadelta T cells. In addition, ESAT-6 also induces the activation and proliferation of gammadelta T cells. Phenotypic analysis indicates that IFN-gamma-producing gammadelta T cells are mainly effector memory cells with the surface phenotype of CD45RA(-)CD62L(-)CCR7(-). These results were further confirmed by the fact that naive gammadelta T cells from cord blood did not produce IFN-gamma in response to ESAT-6. Further studies indicated that stimulation with ESAT-6 directly induced purified gammadelta T cells to produce IFN-gamma, independent of both antigen-presenting cells and CD4(+) T cells. Unexpectedly, depletion of CD4(+) T cells markedly enhanced IFN-gamma production by gammadelta T cells, indicating that CD4(+) T cells regulate the response of gammadelta T cells. Importantly, CD4(+)CD25(+) T regulatory (Treg) cells but not CD4(+)CD25(-) T cells significantly inhibited IFN-gamma production by gammadelta T cells. Taken together, these data demonstrate for the first time that Treg cells can play an important role in the regulation of immune responses of antigen-specific human memory gammadelta T cells.