Abstract Endothelial progenitor cells (EPCs) can be isolated from adult peripheral and umbilical cord blood. EPCs are thought to originate from bone marrow, circulate in peripheral blood, and migrate to sites of angiogenesis. However, the number of circulating EPCs in peripheral blood is remarkably low, and recent genetic studies show that the contribution of bone marrow derived EPCs to newly formed vascular networks is minimal. Further, while endothelial cells (ECs) derived from vessel walls are widely considered to be differentiated mature ECs, these cells retain extensive proliferative potential and can be passaged for at least 40 population doubling in vitro. Based on these observations, we tested whether EPCs potentially reside in vessel walls utilizing a newly developed single cell deposition assay (Blood, 2004). Analogous to a paradigm established in the hematopoietic cell system, we can utilize this assay to reproducibly identify the following EPCs: (1) high proliferative potential - endothelial colony forming cells (HPP-ECFC), which form macroscopic colonies that form secondary and tertiary colonies upon replating, (2) low proliferative potential - endothelial colony forming cells (LPP-ECFC), which form colonies greater than 50 cells, but do not form secondary colonies upon replating, (3) endothelial cell clusters (EC-clusters) that contain less than 50 cells, and (4) mature terminally differentiated endothelial cells (EC), which do not divide. Utilizing this assay, we compared the clonogenic potential of 1000 single adult human dermal microvascular endothelial cells (HMVECds), human umbilical vein endothelial cells (HUVECs), human umbilical artery endothelial cells (HUAECs), human coronary artery endothelial cells (HCAECs), and human aortic endothelial cells (HAECs) to the potential of adult peripheral and umbilical cord blood derived EPCs. We conducted four independent experiments. Remarkably, we demonstrate that a complete hierarchy of EPCs can be identified in EC populations derived from every vessel wall tested (Table I and n=4). Further, we show that ECs derived from each vessel wall cell population tested contain more proliferative EPCs (LPP-ECFCs and HPP-ECFCs) compared to EPCs derived from adult peripheral blood. Percent of 1,000 Single Cells Plated Mature EC EC-Cluster LPP-ECFC HPP-ECFC HUVEC 42±6 18±2 29±9 11±5 HAEC 37±3 23±8 21±4 20±6 HMVECd 65±9 21±6 12±4 2±0.6 HCAEC 46±2 18±2 20±2 16±2 HUAEC 41±1 10±1 27±4 21±2 Adult EPC 81±9 9±1 12±8 0.2±0.2 Cord EPC 50±20 7±2 20±10 23±9 Thus, this study provides evidence that a diversity of EPCs exists in human vessels and provides a new conceptual framework for determining both the origin and function of EPCs in maintaining vessel integrity and contributing to new sites of angiogenesis.