Although hyaluronan (HA), a high molecular weight glycosaminoglycan, is generally believed to regulate tumor growth, invasion, and metastasis, functional roles of HA have only been speculated indirectly from the outcome of blocking HA receptors (e.g., CD44). Using a phage display technique, we recently developed a synthetic peptide (GAHWQFNALTVR; Pep-1) that binds to and inhibits the function of HA. In this study, we have used Pep-1 to determine whether HA directly regulates the behavior of tumor cells. B16-F10 melanoma cells, which constitutively expressed CD44, showed significant adhesion to HA-coated plates, and this adhesion was blocked almost completely either by neutralizing antibodies against CD44 or by Pep-1. These results imply that CD44 is the primary HA receptor mediating the adhesive interaction of the melanoma cells with HA substrates. In contrast, Pep-1 failed to inhibit in vitro proliferation of B16-F10 melanoma cells or the in vitro growth of the cells after s.c. inoculation in mice. Importantly, single injection of Pep-1 significantly reduced the incidence of lung metastasis of i.v. inoculated melanoma cells and prolonged the survival of the tumor-bearing animals. These results suggest a direct contribution of HA to one or more steps in the initial seeding of melanoma cells in the lung microenvironment. Our observations also introduce a new concept that synthetic inhibitors of HA may represent unique tools for studying the roles of HA in tumor biology and perhaps a new class of therapeutic reagents.