Primary human vascular endothelial cells were immortalized by the integration of a single DNA copy of an amphotrophic, replication-deficient retrovirus containing the E6/E7 genes of human papilloma virus. To date, the resulting cell lines, designated EC-RF7 and EC-RF24, have been cultured for more than 1 year. The cell lines have retained a diploid karyotype, display no abnormalities, and are able to grow in a polar mode. Analysis of the EC-RF cell lines by indirect immunofluorescence, using an extensive panel of monoclonal antibodies, showed expression of endothelial cell-specific soluble (von Willebrand factor) and surface-bound antigens (endoglin, PCAM-1) indistinguishable from that of primary cells. In addition, the expression of the markers CD9, 13, 14, 29, 36, 40, 51, and 55 that are not restricted to endothelial cells was also similar for the immortalized and the primary endothelial cells. Immortalization did not alter the expression of the surface adhesion molecules E-selectin, VCAM-1, and ICAM-1 nor transmigration of neutrophils. The regulation of extracellular proteolytic activity by EC-RF24 was established by measuring both the induction of functional tissue factor (promotion of Factor Xa generation) and the functional deposition of plasminogen activator inhibitor 1 in the subendothelial matrix (SDS-resistant complex formation with thrombin). Finally, the biosynthesis of the endothelial cell-specific von Willebrand factor was studied in detail in the EC-RF24 cell line and the results were compared with those of primary endothelial cells.