A number of human endothelial cell lines from umbilical cord cells (HUVECs) have been generated by transfection with SV40 large T and small t antigen sequences. Comparison of these lines with primary cultures of HUVECs has been carried out by monitoring the expression of a number of endothelial cell markers with specific regard to cell age. The secreted levels of the protein plasminogen activator inhibitor (PAI) was found to be significantly reduced in SV40-transfected cells when compared to untransfected controls. Tissue plasminogen activator (tPA) and urokinase (uPA) levels were unchanged. As cells entered crisis, there was a rapid and significant increase in the levels of tPA, uPA, and PAI and this was observed for all clones screened. The endothelial cell marker von Willebrand Factor (vWF) was found intracellularly and was also secreted into the medium. The levels were not altered between transfected and untransfected cells. Angiotensin converting enzyme (ACE) activity was maintained in cell lines at levels found in nonimmortalized HUVECs. Both isoforms (α and β) of IL-1 (interleukin-1) increased as cells approached crisis, and the presence of these cytokines may be responsible for the increased levels of tPA, PAI, and uPA. With one exception, the ability of the transfected cells to produce prostacyclin (PGI₂) was lost by all clones.