A predominant species of heparan sulfate proteoglycan that consisted of at least two subunits linked by disulfide bonding was isolated from cell layers of normal ('cobblestone') bovine vascular endothelial cells in culture. Treatment of the parent molecules with dithiothreitol caused their complete cleavage and permitted the subsequent separation of the larger and smaller subunits on Sepharose CL-4B columns. Removal of dithiothreitol by dialysis resulted in the reformation of large disulfide-bonded molecules but such recombination of the subunits was prevented by prior reductive alkylation using iodoacetamide. Buoyant density gradient analysis as well as gel chromatography on Sepharose CL-6B columns, following alkaline borohydride and nitrous acid treatment of individual carbohydrate-rich subunits, showed that the latter consisted of core proteins associated solely with heparan sulfate glycosaminoglycans. The sizes of the latter were estimated by chromatographic techniques to be approximately 50 kDa in the case of the larger and 14 kDa for the smaller subunit. This is the first description of disulfide-bonded proteoheparan sulfates in endothelial cells.