Suramin is a polysulfonated drug with several biological activities including inhibition of binding of some growth factors to cells, inhibition of tumor cell growth, and of glycosaminoglycan metabolism. We report here that suramin also inhibits binding of the adhesive glycoproteins, thrombospondin and laminin, to immobilized sulfatide with 50% inhibitory doses of 220 and 470 micrograms/ml, respectively. Sulfated glycoconjugates on melanoma cells mediate spreading on thrombospondin by binding to the amino-terminal heparin- and sulfatide-binding domain. This domain is also required for chemotaxis on thrombospondin. We therefore examined the effect of suramin on human melanoma cell spreading and migration. Suramin at 50-400 micrograms/ml specifically inhibited G361 melanoma cell spreading on thrombospondin without affecting cell attachment. Suramin also inhibited spreading of A2058 melanoma cells on thrombospondin and laminin and partially inhibited cell attachment. However, suramin had no effect on G361 or A2058 cell attachment or spreading on fibronectin. Chemotaxis of A2058 and G361 melanoma cells to thrombospondin and laminin were also specifically inhibited by suramin, as was haptotaxis of A2058 melanoma cells to laminin. However, suramin only weakly inhibited haptotaxis of G361 melanoma cells to thrombospondin, which is not mediated by the amino-terminal domain, and did not inhibit haptotaxis to fibronectin. These results suggest a new mechanism for the observed antitumor activity of suramin based on its ability to inhibit interactions of tumor cells with laminin or thrombospondin in the extracellular matrix.