Calf aorta endothelial cells, obtained by collagenase treatment of vessels from freshly killed animals, were cultured in medium 199 supplemented with fetal bovine serum, amino acids, and vitamins. The material released from the vessel wall after collagenase treatment consists of clumps of cells which quickly attach to the dish and spread to form confluent islands of cells. These islands of cells coalesce to form a confluent monolayer in 6 to 8 days. Cultures labeled with [3H]thymidine show an increase in labeling commencing 3 days after initial culture. Cells within monolayers do not overgrow one another and retain their epithelioid appearance after subculture. Identification of endothelial cells was based on culture morphology and by the presence of factor VIII antigen as localized by indirect immunofluorescence microscopy. These data indicate that large numbers of endothelial cells (5 to 7 x 10(6) cells per aorta) can be obtained and maintained in culture.