Abstract: Objective: To investigate the methods of isolating and culturing endothelial progenitor cells (EPCs) from adult peripheral blood mononuclear cells, and study the biological properties of EPCs. Methods: Mononuclear cells were isolated by density-gradient centrifugation and incubated onto fibronectin-coated dishes in endothelial medium in the presence of vascular endothelial growth factor. After cultivation, cells were sequential replanted by allowing cells to adhere to tissue culture dishes during two successive 2-houre incubations in order to remove nonadherent cells. Medium was changed every other days, and the number of early colonies was counted at 7 days after plating. Furthermore, every samples were divided into 2 aliquots and cells from one aliquot were cultivated continually until the late colonies generated. At the same time, another aliquot was handled when the early colonies can be observed after 7 days cultivation. Flow cytometry analysis was used to evaluate cells surface antigen expression and the concentration of vascular endothelial growth factor (VEGF) in cultivation medium was determined with immunosorbent assay (ELISA) method, and the capacity of in vitro vascular genesis was assessed by plating cells onto collagen gels. Results: Early colonies were identified as spindle like sprouting cells radiating from a central core of round cells. When replanted, the early colonies failed to form second colony, and were devoid of the capacity of in vitro vascular genesis. Furthermore, cells derived from the early colonies expressed mainly CD14 and CD45, and concentrations of VEGF in conditioned medium originated from early colonies were increased obviously. In contrasted to early colonies, the late colonies emerged until 21 to 28 days after cultivation and exhibited typically endothelial cells properties. Remarkably, cells originated from late colonies had the ability to form second endothelial colonies after replanted and generated tube-like structures when seeded onto collagen gels. In addition to the clearly morphological differences, cells derived from late clones expressed obviously increased CD146 (P<0.01) and reduced CD45 and CD14 (P<0.001). Conclusions: Under endothelial cultivating conditions, human peripheral blood mononuclear cells can generate early and late colonies. The great majority of cells derived from early colonies belongs to monocyte linage and can secrete VEGF, but has not the ability to differentiate into endothelial cells. Alternatively, cells originated from late colonies exhibit morphologically and biologically characteristics of EPCs.