Tissue hypoxia plays a critical role in normal development, including cardiogenesis. Previously, we showed that oxygen concentration, as assessed by the hypoxia indicator EF5, is lowest in the outflow tract (OFT) myocardium of the developing chicken heart and may be regulating events in OFT morphogenesis. In this study, we identified additional areas of the embryonic chicken heart that were intensely positive for EF5 within the myocardium in discrete regions of the atrial wall and the interventricular septum (IVS). The region of the IVS that is EF5-positive includes a portion of the developing central conduction system identified by HNK-1 co-immunostaining. The EF5 positive tissues were also specifically positive for nuclear-localized hypoxia inducible factor 1α (HIF-1α), the oxygen-sensitive component of the hypoxia inducible factor 1 (HIF-1) heterodimer. The pattern of the most intensely EF5-stained myocardial regions of the atria and IVS resemble the pattern of the major coronary vessels that form in later stages within or immediately adjacent to these particular regions. These vessels include the sinoatrial nodal artery that is a branch of the right coronary artery within the atrial wall and the anterior/posterior interventricular vessels of the IVS. These findings indicate that a portion of the developing central conduction system and the patterning of coronary vessels may be subject to a level of regulation that is dependent on differential oxygen concentration within cardiac tissues and subsequent HIF-1 regulation of gene expression. Developmental Dynamics 235:115–123, 2006. © 2005 Wiley-Liss, Inc.