The outflow tract (OFT) is abnormal in many congenital heart defects. One critical mechanism for morphogenesis of this complex structure is apoptosis. Chicken embryos (stages 19–38; ED4–10) stained with a fluorescent supravital lysosomal dye (LysoTracker Red; LTR) revealed the three-dimensional relationship between structural changes and apoptosis. The LTR staining peaked in the OFT myocardium at stages 27–32, consistent with our previous analyses using other apoptosis assays. While LTR stained under both the pulmonary artery and the aorta, it was most prevalent in the subaortic myocardium before its elimination. Furthermore, LTR staining was most abundant in the myocardium under intensely cytokeratin-positive, thick epicardium. These data support the hypothesis that temporally and spatially restricted apoptosis in the OFT myocardium allows the aorta and pulmonary artery to dock at the appropriate angle and level with the proper ventricle. These data also support a relationship between the differentiating epicardium and cardiomyocyte apoptosis. Developmental Dynamics 229:489–499, 2004. © 2004 Wiley-Liss, Inc.