The isolation of endothelial progenitor cells from human peripheral blood generates a great hope in vascular tissue engineering because of particular benefit when compared with mature endothelial cells. We explored the capability of progenitor-derived endothelial cells (PDECs) to line fibrin and collagen scaffolds in comparison with human saphenous and umbilical cord vein endothelial cells (HSVECs and HUVECs): (a) in a static situation, allowing definition of the optimal cell culture conditions with different media and cell-seeding densities to check cell behaviour; (b) under shear stress conditions (flow chambers or tubular vascular constructs), allowing investigation of cell response and mRNA expression on both substrates by oligonucleotide microarray analysis and quantitative real-time PCR. Well characterized PDECs: (a) could not be expanded adequately with the usual mature ECs culture media; (b) were able to colonize and grow on fibrin glue; (c) exhibited higher resistance to oxidative stress than HSVECs and HUVECs; (d) withstood physiological shear stress when lining both substrates in flow chambers, and their gene expression was regulated; (e) colonized a collagen-impregnated vascular prosthesis and were able to sense mechanical forces. Our results provide an improved qualification of PDECs for vascular tissue engineering.