Objective: The molecular mechanisms that regulate the formation of the conduction system are poorly understood. We studied the developmental expression pattern and functional aspects of the T-box transcription factor Tbx3, a novel marker for the murine central conduction system (CCS). Methods: The patterns of expression of Tbx3, and of Cx40, Cx43, and Nppa, which are markers for atrial and ventricular chamber-type myocardium in the developing heart, were analyzed in mice by in situ hybridization and three-dimensional reconstruction analysis. The function of Tbx3 in regulating Nppa and Cx40 promoter activity was studied in vitro. Results: In the formed heart, Tbx3 is expressed in the sinoatrial node (SAN), atrioventricular node (AVN), bundle and proximal bundle branches (BBs), as well as the internodal regions and the atrioventricular region. Throughout cardiac development, Tbx3 is expressed in an uninterrupted myocardial domain that extends from the sinoatrial node to the atrioventricular region. This expression domain is present in the looping heart tube from E8.5 onwards. Expression of the chamber-type myocardial markers is specifically absent from the Tbx3 expression domain. Tbx3 is able to repress Nppa and Cx40 promoter activity and abolish the synergistic activation of the Nppa promoter by Tbx5 and Nkx2.5. Conclusion: We identified the T-box transcription factor Tbx3 as a novel and accurate marker for the central conduction system. Our analysis implicates a role for Tbx3 in repressing a chamber-specific program of gene expression in regions from which the components of the central conduction system are subsequently formed.