The identification of a specific required carbohydrate structure for the antithrombin III binding site on heparin suggests that there may be specific structures in glycosaminoglycan chains which are necessary for other vascular functions of these carbohydrates. Determining that such differences exist requires a mechanism to isolate heparan sulfates from endothelial cells of specific vascular beds. The present report indicates that cultured venous and aortic endothelial cells synthesize heparan sulfate chains differing in charge density. There are two important conclusions from this work. (i) Endothelial cells from different blood vessels (i.e., vena cava and thoracic aorta) synthesize heparan sulfates which differ in negative charge and sulfation pattern. Specifically, aortic endothelial heparan sulfates have a higher negative charge than venous heparan sulfates. Differences are also observed in the nitrous acid degradation products of the heparan sulfates. (ii) Endothelial cells in culture retain the ability to synthesize different heparan sulfates in vitro after months of subculture under defined conditions. These results indicate that it is feasible to characterize heparan sulfates using cultured endothelial cells from a variety of vascular beds.