T-box (Tbx) genes represent a phylogenetically conserved family of transcription factors that play important roles during embryonic development. Tbx family members have been shown to either activate or inhibit gene expression. However, little is known about the domains within Tbx proteins responsible for mediating gene transcription. While Tbx2 is known to repress gene expression, the domain(s) within Tbx2 remains poorly defined. Deletion of the carboxy-terminus of Tbx2, which contains a domain that is highly conserved with Tbx3 and ET, which has been demonstrated to contain a repression domain, only minimally diminishes the ability of Tbx2 to repress gene expression. However, in combination with the carboxy-terminal truncation, deletion of the amino acids located amino-terminal to the T-box abolished the ability of Tbx2 to repress gene expression. Both of these domains were capable of repressing gene expression when linked to the GAL4 DNA binding domain. In contrast to these two repression domains, the T-box was capable of weakly activating gene expression depending on the promoter context. Deletion analysis of the T-box suggests that this activation domain is located in the amino-terminal end of the T-box. These results reveal a novel transcription repression domain, confirm the presence of a previously implicated domain, and suggest a novel role for the T-box. Taken together, these results provide the basis for understanding the molecular mechanism whereby Tbx2 regulates gene expression and subsequently controls embryonic development.