The plasminogen activator system has been implicated in the modulation of the response to vascular injury. Although urokinase-type plasminogen activator (uPA) and its receptor (uPAR) may enhance matrix degradation as well as migration and invasion by smooth muscle cells (SMCs), their roles in cell adhesion are uncertain. Therefore, we examined the ability of uPA and uPAR to modulate adhesion of cultured human vascular SMCs to various matrices. We demonstrated a dose-dependent stimulation of adhesion by single-chain uPA (scuPA) to vitronectin (maximum 1.55-fold [+/-0. 04-fold] increase, 10 nmol/L, P<0.002) but not to laminin, collagen I, or collagen IV. Baseline adhesion to vitronectin was completely inhibited by both EDTA and RGD peptide but was restored to >40% of control in the presence of scuPA (P=0.001 and 0.046, respectively). Adhesion to vitronectin was also significantly enhanced by the amino-terminal fragment of uPA (P=0.007) and two-chain, high-molecular-weight uPA (P<0.01) but not by the low-molecular-weight fragment of uPA, which lacks the receptor-binding domain. Aprotinin, a plasmin inhibitor, had no effect on baseline or scuPA-stimulated adhesion, suggesting a plasmin-independent process. Preincubation of scuPA with soluble uPAR inhibited scuPA stimulation of adhesion by 88+/-14% (P=0.01), as did pretreatment of SMCs with phosphatidylinositol-specific phospholipase C, which removes glycophosphatidylinositol-anchored proteins, including uPAR. Antibodies to both alphavbeta3 and alphavbeta5 integrin inhibited baseline adhesion but not scuPA stimulation. Finally, coating plates with scuPA alone enabled cell adhesion, which could be inhibited by both soluble uPAR and anti-uPAR antibodies. These data suggest that uPA stimulates adhesion of SMCs specifically to vitronectin and that it is mediated by an interaction with uPAR. Upregulation of both proteins after vascular injury may facilitate migration through stimulation of both matrix degradation and cell adhesion.