CD44 represents a family of glycoproteins that are present on the surfaces of some types of lymphocytes, macrophages, and epithelial cells. In the present study, we have found that CD44 is also present on murine megakaryocytes and peripheral blood platelets as judged by immunohistochemical staining. Western blotting of platelet proteins indicated that this CD44 was predominantly of the 85-kD form. This form of CD44 also had the capacity to bind hyaluronan, because detergent extracts of platelets as well as intact platelets could bind soluble [3H]hyaluronan, and this property was blocked by antibodies directed against CD44. More importantly, isolated platelets could attach to the hyaluronan-containing extracellular matrix produced by cultured rat fibrosarcoma cells. This attachment took place in the absence of divalent cations and could be blocked by pretreating the rat fibrosarcoma cells with hyaluronidase or by the addition of an antibody to CD44. These results suggested that CD44 was responsible for the attachment of platelets to hyaluronan. Histochemical staining also showed that hyaluronan was present immediately beneath the endothelial cells of many blood vessels of various tissues, such as the dermis, lamina propria of the intestinal tract, the lungs, and the pericardium. Thus, it is possible that CD44 plays an important role in the attachment of platelets to the surface of exposed connective tissue after injury to endothelial cells.