Notch1 is an important regulator of intercellular interactions in cardiovascular development. We show that the nuclear-localized, cleaved and active form of Notch1, the Notch1 intracellular domain (N1ICD), appeared in mesothelial cells of the pro-epicardium during epicardial formation at looped heart stages. N1ICD was also present in mesothelial cells and mesenchymal cells specifically within the epicardium at sulcus regions. N1ICD-positive endothelial cells were detected within the nascent vessel plexus at the atrioventricular junction and within the compact myocardium (Hamburger and Hamilton stage [HH] 25–HH30). The endothelial cells expressing N1ICD were surrounded by N1ICD-positive smooth muscle cells after coronary orifice formation (HH32–HH35), while N1ICD expression was absent in the mesenchymal and mesothelial cells surrounding mature coronary vessels. We propose that differential activation of the hypoxia/HIF1-VEGF-Notch pathway may play a role in epicardial cell interactions that promote epicardial epithelial/mesenchymal transition and coronary progenitor cell differentiation during epicardial development and coronary vasculogenesis in particularly hypoxic sulcus regions. Developmental Dynamics 238:162–170, 2009. © 2008 Wiley-Liss, Inc.