Mechanically challenged tissue-engineered organs, such as blood vessels, traditionally relied on synthetic or modified biological materials for structural support. In this report, we present a novel approach to tissue-engineered blood vessel (TEBV) production that is based exclusively on the use of cultured human cells, i.e., without any synthetic or exogenous biomaterials. Human vascular smooth muscle cells (SMC) cultured with ascorbic acid produced a cohesive cellular sheet. This sheet was placed around a tubular support to produce the media of the vessel. A similar sheet of human fibroblasts was wrapped around the media to provide the adventitia. After maturation, the tubular support was removed and endothelial cells were seeded in the lumen. This TEBV featured a well-defined, three-layered organization and numerous extracellular matrix proteins, including elastin. In this environment, SMC reexpressed desmin, a differentiation marker known to be lost under standard culture conditions. The endothelium expressed von Willebrand factor, incorporated acetylated LDL, produced PGI2, and strongly inhibited platelet adhesion in vitro. The complete vessel had a burst strength over 2000 mmHg. This is the first completely biological TEBV to display a burst strength comparable to that of human vessels. Short-term grafting experiment in a canine model demonstrated good handling and suturability characteristics. Taken together, these results suggest that this novel technique can produce completely biological vessels fulfilling the fundamental requirements for grafting: high burst strength, positive surgical handling, and a functional endothelium.