Rationale: The ventricular conduction system controls the propagation of electric activity through the heart to coordinate cardiac contraction. This system is composed of specialized cardiomyocytes organized in defined structures including central components and a peripheral Purkinje fiber network. How the mammalian ventricular conduction system is established during development remains controversial. Objective: To define the lineage relationship between cells of the murine ventricular conduction system and surrounding working myocytes. Methods and Results: A retrospective clonal analysis using the ␣-cardiac actin nlaacZ/؉ mouse line was carried out in three week old hearts. Clusters of clonally related myocytes were screened for conductive cells using Key Words: conduction system Ⅲ cell lineage Ⅲ clonal analysis Ⅲ nlaacZ Ⅲ Purkinje fibers T he cardiac conduction system controls the generation and propagation of electric activity through the heart to coordinate cardiac contraction. Atrial contraction is initiated by the sinoatrial node or pacemaker. After a delay mediated by the atrioventricular node (AVN), the ventricular conduction system (VCS) ensures rapid propagation of electric activity to the ventricular apex. 1 The VCS is comprised of a central component, the atrioventricular (AV or His) bundle and right and left bundle branches, and a peripheral component composed of a dense ellipsoid network of Purkinje fibers. These structures have been well characterized in adult mouse and human hearts by their specific histological and electrophysiological properties. 2,3 However, despite the clinical importance of the VCS in regulating cardiac rhythm, important questions remain as to the origin and the mode of development of the mammalian VCS. 4 Existing views of VCS development are largely based on data obtained in avian embryos, despite major anatomic differences within the VCS between birds and mammals. In the avian system, lineage analysis using replication defective retroviral labeling has demonstrated that conductive cells share common progenitors with working cardiomyocytes and that the VCS develops by a process of induction and recruitment of myocytes through endothelial derived signals. 5,6 According to this model, conductive myocytes are nonproliferative and subsequent growth of the conduction system occurs by accretional recruitment of new myocytes (or ingrowth). However, in the chick, a perivascular network of Purkinje fibers is localized deep in the myocardium in proximity to coronary vessels, whereas in the mouse, as in humans, Purkinje fibers are present only at the subendocardial ventricular surface 1,7,8 and it is unclear whether the chick model is applicable to the mammalian VCS. In mammals an outgrowth model has been proposed for development of the central VCS, by which conductive cells develop independently of the working myocardium from a pool of conductive Original