Tumor cell migration and invasion require complex interactions between tumor cells and the surrounding extracellular matrix. These interactions are modified by cell adhesion receptors, as well as by proteolytic enzymes and their receptors. Here, we study the influence of the protease urokinasetype plasminogen activator (uPA) and its receptor (uPAR) on melanoma cell adhesion to, and migration on, the extracellular matrix protein vitronectin (VN). Cell adhesion to VN, but not to type I collagen, is significantly enhanced in the presence of either uPA or its amino-terminal fragment (ATF). Soluble uPAR can inhibit this effect, indicating that uPA/ uPAR on melanoma cells can function as a VN receptor. In the absence of bivalent cations, uPA/uPAR can promote cell attachment on VN, but not cell spreading, suggesting that the glycosylphosphatidylinositol (GPI)-anchored uPAR alone is unable to organize the cytoskeleton. Chemotactic melanoma cell migration on a uniform VN matrix is inhibited by uPA and ATF, implying that cell motility decreases when uPA/uPAR acts as a VN receptor. In contrast, plasminogen activator inhibitor 1 (PAI-1) can stimulate melanoma cell migration on VN, presumably by inhibiting uPA/uPAR-mediated cell adhesion to VN and thereby releasing the inhibition of cell migration induced by uPA. Together, our data implicate components of the plasminogen activation system in the direct regulation of cell adhesion and migration, thereby modulating the behavior of malignant tumor cells.