Cell death is thought to play an important role in mammalian cardiogenesis, although a precise map of its distribution during the crucial period of cardiac septation has so far been lacking. In this study, the spatiotemporal distribution of programmed cell death (PCD) during mouse cardiac septation is described between embryonic days 10.5 and 13.5. Two types of foci of cell death can be demonstrated in the developing heart. Those with high-intensity, with a PCD index greater than 1%, are clearly visible on individual TUNEL-assayed sections. Low-intensity foci, with a PCD index of less than 1%, become visible only following summation of data. High-intensity foci occur exclusively within the endocardial cushions of the outflow tract and atrioventricular region, appearing at the 52–54 somite stage (late E11.5), concomitant with the formation of the central mesenchymal mass. Low-intensity foci are present throughout the period of cardiac development from E10.5 to E13.5 and are frequently localized to regions of septation, such as the muscular ventricular septum and the mesenchymal cap of the primary atrial septum. Expression of Fas and FasL corresponds to these low-intensity foci, but not those with high-intensity, suggesting that activation of this death receptor may be specifically involved in molecular control of the low-intensity foci. Anat Rec Part A 277A:355–369, 2004. © 2004 Wiley-Liss, Inc.