This chapter examines the different aspects of human endothelial cells in vitro. Arterial vascular disease accounts for substantial morbidity and mortality in humans. In many of these conditions, endothelial injury has been postulated to be an important component. There have been numerous attempts to propagate endothelial cells in vitro to study suspected pathogenic factors in a systematic fashion under conditions where individual variables can be manipulated. The identification of cells in vitro is often difficult, depending on the maintenance of in vivo functional and/or morphological traits. An additional feature characteristic of endothelial cells is the presence of the cell surface enzyme angiotensin-converting enzyme. Human endothelial cells, like all mammalian cells, require plasma or serum for continued proliferation. In serum-free media at sparse density, the cells become quiescent and do not proliferate. If endothelial cells produce a mitogen, consideration of the relationships of endothelial cells and smooth muscle cells in vivo becomes considerably more complex.