The T-box transcription factor T-bet has been reported to augment the activity of IFN-gamma reporter constructs and to be required for CD4(+), but not CD8(+), T cell production for IFN-gamma. Despite these observations, the precise sequence targets of T-bet within the IFN-gamma locus have not been identified and the nature of T-bet's role in selectively augmenting IFN-gamma production in CD4(+) T cells has not been elucidated. As an initial step in this process, we examined the basis of T-bet-dependent augmentation of IFN-gamma reporter constructs to identify specific targets of this factor within the IFN-gamma locus. Deletion of previously proposed TDB and TRU elements left T-bet-induced IFN-gamma reporter activity unchanged, suggesting the existence of additional T-bet-responsive elements. We identified several additional monomeric Brachyury consensus elements within the proximal IFN-gamma promoter that operate cooperatively to increase both constitutive and stimulated promoter activity. The most proximal of these Brachyury elements is most significant quantitatively in mediating T-bet-dependent promoter augmentation. Mutation of this with any of the other Brachyury elements leads to a near eradication of T-bet-dependent promoter activation. The identification of these individual monomeric Brachyury-binding sites within the IFN-gamma locus should facilitate the in vivo analysis of the function of T-bet in the lineage- and background-dependent requirement for T-bet in IFN-gamma gene regulation.