Abstract Endothelial cells (EC) were harvested by 0.1% collagenase treatment for adult human thoracic aortas obtained 1–3 h after sudden death. At least 35–70% of EC were removed from the intimal surface of aorta, 90–95% of them being viable. Plating efficiency was 70–80%. Monolayer formation was achieved at a seeding density of 5–8 × 102 cells/mm 2. The cells were identified as endothelium by the presence of Factor VIII antigen, Weibel-Palade bodies and typically endothelial morphology at confluence. Unlike endothelial cultures derived from human umbilical veins and infant aortas, primary cultures obtained from human adult aortas contain multinuclear EC with Factor VIII antigen and Weibel-Palade bodies. The number of multinuclear EC in cultures isolated from aortas affected by atherosclerosis was 2-fold higher ( P < 0.05) than in cultures obtained from grossly normal aortas taken from donors of the same age. EC with numerous lipid inclusions revealed by oil-red-O staining were present in all the EC primary cultures derived from aortas affected by atherosclerosis. No oil-red-O-positive cells were detected among the EC cultured from infant aorta, aorta of young donors, and umbilical yein. An electron microscopic examination of EC from atherosclerotic aorta in culture and in situ failed to reveal any ultrastructural peculiarities distinguishing multinuclear EC from the mononuclear EC.