We developed a new device, the vein holder, to improve yield and purity of enzymatic harvests of venous endothelial cells. External jugular veins of mongrel dogs were dissected by a no-touch technique. In vitro length and circumference of the vein segments were decreased to about half of the in situ dimensions. The vein holder enabled mounting of the veins at 80% of their in situ length during endothelial cell harvesting. Trypan blue staining and scanning electron microscopic observations revealed that vein eversion as well as the new vein holder technique successfully removed the endothelium. Endothelial cell harvests by the eversion technique were, however, low and varied in size, viability, and purity. In contrast, the defined handling by the new vein holder technique regularly provided markedly increased amounts of endothelial cells. Most of the cells attached and developed cultures consisting of endothelial cells only, as shown by the uptake of DilAcLDL. Prostacyclin production of confluent cultures was similar to that of native veins. It is concluded that minimal handling, defined mounting, and prevention of overfilling the vein markedly improves endothelial cell harvests, providing greater amounts of viable and purified endothelial cells.