We analyzed a cryopreservation protocol which improves long-term storage of endothelial cells (EC) for tissue engineering purposes. Human umbilical vein EC were frozen in a high-potassium solution containing 10% dimethyl sulfoxide using 3 different cooling rates. After a storage time in liquid nitrogen of 1, 4, or 12 months, samples were thawed and compared to fresh cells in terms of growth rates, anti-inflammatory, and anticoagulant functions. Independent of cooling rate and storage time, the retrieval after cryopreservation ranged between 60% and 80%. However, viability of the cells cryopreserved at 10°C/min decreased significantly from 78 ± 5% to 64 ±3% with storage. Storage time of 4 months resulted in a decreased cell multiplication factor over 4 and 12 days in culture. The lag phases returned to normal in the next passage. Thawed cells showed increased metabolic activity, reduced expression of thrombomodulin, and unchanged basal expression of adhesion molecules. However, the tumor necrosis factor–induced expression of adhesion molecules was significantly increased after long-term storage. This effect was partially compensated after expansion of the cells, whereas the prostacyclin release increased. Expansion of cryopreserved/ thawed EC resulted in highly proliferative cells with antithrombotic properties and a capacity for inflammatory reactions, which makes them suitable for vascular tissue engineering.