The vascular smooth muscle cells of coronary arteries are distinguished from those of the proximal aorta by a number of structural and functional criteria which may include an increased propensity for atherosclerotic transformation. At present, the source of this variation between smooth muscle subpopulations is uncertain. Whilst smooth muscle of the proximal aorta is thought to be derived from neural crest, the origin of coronary vascular smooth muscle remains uncharacterized. We have previously shown that precursors of the coronary vasculature enter the tubular heart on the same day as the epicardial mantle starts to envelop the myocardium and that coronary vessels form by ingrowth of these migratory precursors and not by outgrowth from the aorta (Mikawa and Fischman, 1992). To study the origin of coronary smooth muscle cells, the proepicardial organ, from which epicardial cells arise, was tagged with either a vital dye (DiI) or replication-defective retroviruses encoding beta-galactosidase. Cellular lineage marking was achieved by either direct targeting of putative vasculogenic cells in the proepicardium in ovo or tagging dissected proepicardial cells in vitro followed by transplantation to stage-matched host embryos. Monitoring of tagged cells during coronary vasculogenesis indicate incorporation of proepicardial-derived cells into three vessel-associated populations; coronary smooth muscle, perivascular connective tissue, and endothelial cells. Immunoconfocal microscopy identified both endothelial and smooth muscle cell populations within the proepicardial organ. The results demonstrate that: (1) the proepicardium contains a progenitor population of coronary smooth muscle cells that migrates into the heart along with ingrowth of the epicardium and (2) prior to the migration, the coronary smooth muscle lineage is established.