Tissue culture of retinal vessels from fetal calf eyes produced colonies of endothelium, pericytes, and smooth muscle. Identification of endothelial cells was based upon culture morphology, [3H]thymidine labeling of isolated vessels, and factor VIII immunofluorescence. Thimerosal added to the culture medium destroyed pericytes and muscle cells, leaving only endothelial cell colonies in the primary cultures. This tissue culture system may be useful in the study of retinal vascular cell biochemistry and pathophysiology.