A functional pacemaking/conduction system is essential for maintaining normal cardiac function. However, no reproducible model system exists for studying the specialized cardiac pacemaking/conduction system in vitro. Although several molecular markers have been shown to delineate components of the cardiac conduction system in vivo, the functional characteristics of the cells expressing these markers remains unknown. The ability to accurately identify cells that function as cardiac pacemaking cells is crucial for being able to study their molecular phenotype. In differentiating murine embryonic stem cells, we demonstrate the development of an organized cardiac pacemaking/conduction system in vitro using the co-expression of the minK-lacZ transgene and the chicken GATA6 (cGATA6) enhancer. These markers identify clusters of pacemaking "nodes" that are functionally coupled with adjacent contracting regions. cGATA6-positive cell clusters spontaneously depolarize, emitting calcium signals to surrounding contracting regions. Physically separating cGATA6-positive cells from nearby contracting regions reduces the rate of spontaneous contraction or abolishes them altogether. cGATA6/minK co-positive cells isolated from EBs display characteristics of specialized pacemaking/conducting cardiac myocytes with regard to morphology, action potential waveform, and expression of a hyperpolarization-activated depolarizing current. Using the cGATA6 enhancer, we have isolated cells that exhibit electrophysiological and genetic properties of cardiac pacemaking myocytes. Using molecular markers, we have generated a novel model system that can be used to study the functional properties of an organized pacemaking-conducting-contracting system in vitro. Moreover, we have used a molecular marker to isolate a renewable population of cells that exhibit characteristics of cardiac pacemaking myocytes.