We investigated the effects of native, oversulfated, and desulfated fucoidans and heparin on the invasion of 3 LL cells through Matrigel. Of the four polysaccharides tested, oversulfated fucoidan was the most potent inhibitor of tumor cell invasion and inhibited most potently and specifically the tumor cell adhesion to laminin. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the binding of elastase-cleaved laminin to fucoidan- and heparin-Sepharoses showed that both polysaccharides bound to the 62 and 56 kDa fragments. Pretreatment of 3LL cells with native or oversulfated fucoidan reduced their adhesive potency to laminin. The two fucoidans inhibited further the laminin binding of 3 LL cells which had been pretreated with a laminin-based pentapeptide, YIGSR. These results suggest that fucoidan specifically binds to not only the heparin binding domain(s) of laminin but also site(s) other than the cell surface laminin receptor. 3 LL cells secreted a 50 kDa form of urokinase-type plasminogen activator (u-PA). The extracellular level of u-PA activity was increased 1.7 times by addition of laminin but not type IV collagen. Oversulfated fucoidan most potently reduced the increased u-PA levels. Therefore, the reduction in in vitro invasiveness of 3 LL cells in response to either fucoidan or its oversulfated derivative may result from an inhibition of physical interaction between the tumor cells and the Matrigel (laminin), followed by a suppression of the laminin-induced increase in extracellular u-PA.