Tbx3, a member of the conserved family of T-box developmental transcription factors, is a transcriptional repressor required during cardiogenesis for the formation and specification of the sinoatrial node, the pacemaker of the heart. Both the TBX3 and the highly related TBX2 genes are also associated with several cancers, most likely as a consequence of their powerful anti-senescence properties mediated via suppression p14(Arf) and p21(CIP) expression. In melanoma, the TBX2 gene is frequently amplified and inhibition of Tbx2 function leads to senescence and up-regulation of p21(CIP), a Tbx2 target gene. Tbx3 + 2a is a splice variant containing an extra 20 amino acids encoded by exon 2a inserted into the highly conserved T-box DNA-binding domain. We find here that Tbx3 + 2a is evolutionary conserved and that similar insertions are largely absent from the T-box domains of other T-box factors. Tbx3 + 2a has been reported to lack DNA-binding ability and act as a functional antagonist of Tbx3. By contrast, we now demonstrate that both Tbx3 and Tbx3 + 2a bind the consensus T-element, the p21(CIP1) promoter, and the Nppa cardiac target gene. Both isoforms also function as repressors of p21(CIP1) and Nppa promoter activity and interact with homeobox factor Nkx2-5. When ectopically expressed in the embryonic heart of mice, Tbx3 and Tbx3 + 2a both suppressed chamber formation and repressed expression of cardiac chamber markers Nppa and Cx40. The results suggest that in the assays used, Tbx3 and Tbx3 + 2a are functionally equivalent and that like Tbx2, Tbx3 may also function as an anti-senescence factor in melanoma.