Endothelial cells were obtained from white-tailed deer carotid arteries and umbilical vessels by instilling a weak collagenase type II solution. Cell growth was best when cultures were grown in plates coated with either fibronectin or laminin. Several commercially available media supported the growth of these cells when supplemented with a commercially available endothelial growth supplement and 10% fetal calf serum. Cells obtained could be characterized as endothelium by ultrastructural characteristics and by the uptake of fluorescent acetylated low-density lipoprotein. Cells stained positive for factor VIII-related antigen up to passage 2, but staining was inconsistent by passage 3, and no immunoreactivity could be demonstrated after passage 4.