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Introduction: Currently constructed biopacemakers based on single gene transfer strategies function suboptimally, with periods of slow heart rates and instabilities during rest. In the sinoatrial node (SAN), the dominant native pacemaker, multiple genes are required for proper impulse formation and impulse propagation. TBX3 is an important regulator of the SAN gene program during heart development. We examined the effects of inducible TBX3 overexpression in adult hearts and in vitro we explored whether lentiviral TBX3 overexpression may be used in biopacemaker engineering. Methods: In vivo atrial and ventricular expression levels of the connexin isoforms Cx43 and Cx40 (impulse propagation) and SCN5A were studied in mice with tamoxifen inducible overexpression of TBX3 using quantitative PCR analysis. Single neonatal rat cardiac myocytes were transduced with TBX3 expressing lentivirus to analyze the effects of TBX3 on action potentials and membrane currents (impulse formation) using the perforated patch-clamp technique. Results: In vivo, Cx43, Cx40 and SCN5A, which are not or only moderately expressed in the native SAN, were severely down-regulated to 20%, 15%, and 40%, respectively, by TBX3 (n=12; pK1 ), and fast sodium current (I Na ). These properties are typical of SAN pacemaker cells. Conclusions: TBX3 can act as a strong repressor of the working myocardium gene program in the adult heart. Overexpression of TBX3 might be a useful tool in biopacemaker gene and cell therapy.