The life-span in vitro and other proliferative characteristics of a strain of endothelial cells cloned from the aorta of a fetal calf were examined. Cultures of these cells had a replicative life-span of approximately 80 cumulative population doublings. Growth rates in the logarithmic phase and plateau densities decreased as the cumulative population-doubling level increased. After approximately 65 percent of the life-span of a culture was completed, the percentage of cells that incorporated [3H]thymidine during a 24-hour labeling period began to decrease rapidly. The cells expressed factor VIII antigen and their intercellular borders were stainable with silver nitrate throughout the life-span of each culture. Average cellular attachment size increased more than threefold between cumulative population-doubling levels 41 and 80. The facility with which cloned strains of endothelial cells can be isolated should encourage further exploitation of this important cell culture model.