Endothelial cells from a variety of vascular beds can be isolated, purified, grown in culture, passaged, and established as a confluent monolayer on membrane supports. The culture of endothelial cells from porcine pulmonary artery, porcine aorta, porcine coronary arteries, bovine adipose tissue microvessels, human foreskin microvessels, and human umbilical vein is described. Once isolated, these cells can be cultured on membrane supports forming a confluent monolayer. The integrity of the monolayer, its relation to other cells, and its function as a barrier or transport system can be studied. Attention to the details of isolation, purification, and seeding are critical to the establishment of an intact monolayer.