The heparan sulfate proteoglycans that bind and activate antithrombin III (aHSPGs) are synthesized by endothelial cells as well as other nonvascular cells. We determined the amounts of cell surface-associated and soluble aHSPGs generated by the rat fat pad endothelial (RFP) cell line and the fibroblast (LTA) cell line. The RFP cells exhibit higher levels of cell surface-associated aHSPGs as compared to LTA cells, whereas LTA cells release larger amounts of soluble aHSPGs as compared to RFP cells. After confluence RFP cells show an increase in both cell surface-associated and soluble aHSPGs. In contrast, postconfluent LTA cells maintain a constant level of cell surface-associated and soluble aHSPGs. These observations indicate that different cell types can preferentially accumulate aHSPGs as cell surface-associated or soluble forms which could reflect alternate biological functions.