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Smooth muscle cells (SMCs) of the proximal thoracic aorta are embryonically derived from the second heart field (SHF) and cardiac neural crest (CNC). However, distributions of these embryonic origins are not fully defined. The regional distribution of SMCs of different origins is speculated to cause region-specific aortopathies. Therefore, the aim of this study was to determine the distribution of SMCs of SHF and CNC origins in the proximal thoracic aorta.Mice with repressed LacZ in the ROSA26 locus were bred to those expressing Cre controlled by either the Wnt1 or Mef2c (myocyte-specific enhancer factor 2c) promoter to trace CNC- and SHF-derived SMCs, respectively. Thoracic aortas were harvested, and activity of β-galactosidase was determined. Aortas from Wnt1-Cre mice had β-galactosidase–positive areas throughout the region from the proximal ascending aorta to just distal of the subclavian arterial branch. Unexpectedly, β-galactosidase–positive areas in Mef2c-Cre mice extended from the aortic root throughout the ascending aorta. This distribution occurred independent of sex and aging. Cross and sagittal aortic sections demonstrated that CNC-derived cells populated the inner medial aspect of the anterior region of the ascending aorta and transmurally in the media of the posterior region. Interestingly, outer medial cells throughout anterior and posterior ascending aortas were derived from the SHF. β-Galactosidase–positive medial cells of both origins colocalized with an SMC marker, α-actin.Both CNC- and SHF-derived SMCs populate the media throughout the ascending aorta. The outer medial cells of the ascending aorta form a sleeve populated by SHF-derived SMCs.