&NA;Mostin vitrostudies of human endothelial cells have relied on cells derived from human umbilical veins (HUVEC); however, heterogeneity of primary cultured endothelial cells can make critical interpretation of results difficult. Several endothelial cell lines have been produced to serve as a more constant source of endothelial cells. In this study, we characterized the endothelial cell lines EVLB3and EVLC2derived from HUVEC, and EVLK1and EVLK2derived from human iliac vein endothelial cells (HIVEC). These cell lines maintained the typical endothelial cell cobblestone morphology and appeared to be growth factor independent. They lost PECAM‐1 and von Willebrand factor, GP96 was reduced to the level of vascular smooth muscle cells (SMC), but &agr;SMC‐actin was far less than in vascular SMC. Antigen levels of tissue‐type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI‐1) were comparable with young endothelial cells, and mRNA was present for tPA, PAI‐1, tissue factor (TF), tissue factor pathway inhibitor and thrombomodulin. This study revealed that mRNA and protein expression of coagulation and fibrinolytic factors was influenced by the stage of cell confluence. No differences could be detected between the endothelial cell lines derived from HUVEC and HIVEC. These cell lines may be a useful tool for studies on cellular interactions of fibrinolytic components or exploring the regulation of TF expression.