Dermatan sulfate (DS) and heparan sulfate (HS) are the major anticoagulant and antithrombotic glycosaminoglycans (GAGs) found in the vessel walls. However, the exact tissue location and the specific function of these two GAGs under normal and pathological conditions is still unclear. We attempted to investigate the distribution of these two GAGs in different layers of the human umbilical vein. Initially an endothelial cell monolayer was removed from the vessel surface by a mild collagenase digestion, leaving a naked vessel wall without the endothelium. Then, the media layer was removed by a more extensive collagenase digestion, as indicated by a histological examination. GAGs were extracted from the intact vein, before and after removal of the endothelial layer and the media layer. Our data shows that most of the vein HS is in the endothelial cell layer, removed by mild collagenase digestion. Furthermore we assessed the anticoagulant activity of the vein GAGs using a thrombin inhibition assay mediated by antithrombin. The umbilical vein wall, as we remove the endothelial cells by mild and extensive collagenase digestion respectively, the remaining GAGs show a decreased anticoagulant activity, which was ascribed to different populations of HS. In fact, as we determined the anticoagulant activity of the HS from these three populations, we observed the same decrease in the anticoagulant activity described for the total GAG. In contrast, the DS obtained from these different vein wall preparations shows a comparable anticoagulant activity. Overall, these observations indicate  different populations of HS, with distinct anticoagulant activity and distribution along the different layers of the vessel wall. In contrast, a single population of DS is found in the different regions of the vein wall.