Tissue-specific heterogeneity of endothelial cells, both structural and functional, plays a crucial role in physiologic as well as pathologic processes, including inflammation, autoimmune diseases and tumor metastasis. This heterogeneity primarily results from the differential expression of adhesion molecules that are involved in the interactions between endothelium and circulating immune cells or disseminating tumor cells. Among these molecules present on endothelial cells is hyaluronan (HA), a glycosaminoglycan that contributes to primary (rolling) interactions through binding to its main receptor CD44 expressed on leukocytes and tumor cells. While the regulation of CD44 expression and function on either leukocytes or tumor cells has been well characterized, much less is known about the ability of endothelial cells to express HA on their surface. Therefore, in these studies we analyzed HA levels on tissue-specific endothelium. We used endothelial cell lines of different origin, including lung, skin, gut and lymph nodes that had been established previously as model lines to study interactions between the endothelium and leukocytes/tumor cells. Our results indicate that HA is accumulated on the surface of all endothelial cells examined. Moreover, retention of endogenous HA differs between the lines and may depend on their tissue origin. Analysis of binding of exogenous HA reveals the presence of specific HA binding sites on all endothelial cell lines tested. However, the retention of endogenous HA and the binding of exogenous HA is mediated through a CD44-independent mechanism.