Endothelial cell lines are commonly used in in vitro studies to avoid problems associated with the use of primary endothelial cells such as the presence of contaminating cells, the difficulty in obtaining larger numbers of cells, as well as the progressive loss of cell viability and expression of endothelial markers in the course of in vitro propagation. We have analyzed the characteristics defining distinctive endothelial phenotypes in the cell lines EA.hy926, ECV304, EVLC2, HAEND, HMEC-1, ISO-HAS-1 and a cell line recently generated in our laboratory, HPMEC-ST1.6R, and have compared these phenotypes with those found in primary human endothelial cells isolated from umbilical vein (HUVEC), lung (HPMEC), and skin (HDMEC). The analysis revealed significant differences in phenotype expression between primary cells and the cell lines. Constitutive expression of von Willebrand factor, CD31, and CD34 and induced expression of cell adhesion molecules, ICAM-1, VCAM-1, and E-selectin and cytokines, IL-6, IL-8, MCP-1, and GM-CSF on stimulation with proinflammatory stimuli, as well as the uptake of DiI-Ac-LDL and the formation of cord-like structures on Matrigel, were typically observed in the primary cells. However, most cell lines exhibited only a few of these endothelial characteristics. Only HPMEC-ST1.6R exhibited the major constitutive and inducible endothelial cell characteristics and showed an angiogenic response on Matrigel similar to that of primary HPMEC. Thus, HPMEC-ST1.6R will be a valuable in vitro model system in which to study pathomechanisms and angiogenesis of the mature microvascular endothelium in vitro.