A procedure for the isolation and cultivation of endothelium from the marginal vessels of the rabbit ear is described. Endothelial cells, isolated by slow perfusion with a trypsin solution, are cultured in minimal essential medium supplemented with 10% fresh rabbit serum for up to 6 mo. In primary culture, marginal vessel endothelial cells grow in an expanding circular pattern with closely apposed cell membranes. Weibel-Palade bodies, subcellular organelles unique to endothelial cells in situ, are present in both primary and in serially cultivated cells (12 passages). In intact skin, Weibel-Palade (W-P) bodies are observed in the perinuclear cytoplasm in close proximity to the cell membrane facing the vascular lumen. 8-16 tubules of 200 A diameter are present in each body. In primary and subcultured cells, W-P bodies of identical size are seen in the vicinity of the Golgi apparatus and in close proximity to the outer cell membrane. At the optimum serum concentration (10%), a cell doubling time of 72-96 h is observed. When growth in normal rabbit serum and in platelet-poor serum is compared, a slower growth rate is observed in the absence of platelets, suggesting that factors released by platelets affect endothelial cell proliferation. However, addition of crude platelet factor does not substitute for complete serum. Fibroblast growth factor is not mitogenic for rabbit marginal vessel endothelium in vitro.