The cardiac conduction system (CS) controls the generation and propagation of electrical activity through the heart to coordinate cardiac contraction. This system is composed of specialized cardiomyocytes divided in defined structures including the sinoatrial node, the atrio-ventricular node (AVN), the His bundle, bundle branches and peripheral Purkinje fibers. Nevertheless, despite the clinical importance of the conduction system in regulating cardiac rhythm its developmental origin is controversial.A retrospective clonal analysis was performed in order to establish the lineage relationship between cells of the ventricular CS and the surrounding working myocardium. The α-cardiac actinnlaacZ clonal analysis mouse line was crossed with the Cx40-eGFP transgenic line in which cells of the conduction system are readily detectable. Clonally related clusters of β-galactosidase positive myocytes containing GFP positive cells were scored in 3 week old hearts. The presence of hearts with large clones demonstrates that the entire ventricular CS originates from a pool of early progenitors during the dispersive growth phase of cardiac development. The analysis of clonally related clusters reveals the existence of two types of cluster underlying a common mode of development for the different components of the ventricular CS. A first step of differentiation/induction from a common progenitor with contractile cardiomyocytes is followed by proliferation of commited conductive cells. Our data suggest that cells of the central components (AVN, HIS) of the ventricular CS segregate early during development while differentiation of the peripheral CS occurs over an extended period of time. Furthermore, our study reveals different modes of growth for Purkinje fibers in the right and left ventricles that appear to be correlated with the different morphological properties of the ventricles. Our analysis provides new insights into previous controversies existing in the literature concerning the mode of development of the ventricular CS. Detailed understanding of ventricular CS formation is an important issue given that ventricular arrhythmias are major factors of morbidity and mortality and can result from defects in ventricular conduction system development.