Protease nexin-1 (PN-1) and plasminogen activator inhibitor-1 (PAI-1) are serine protease inhibitors that bind to the extracellular matrix protein vitronectin (VN) with high affinity. PAI-1 is known to inhibit cell adhesion and migration by binding to VN and inhibiting the interaction with integrins or the urokinase receptor (uPAR). Unexpectedly, PN-1 was found to increase the association between VN and uPAR in the presence of enzymatically active uPA. Through this mechanism PN-1 also stimulated uPAR-dependent cell adhesion to immobilized VN. In contrast to PAI-1, PN-1 did not influence VN binding to integrins or integrin-mediated cell adhesion. Upon adhesion of monocytes to VN there was an accumulation of uPAR and PN-1 at the interface between the cell and the matrix, whereas on fibronectin (FN) both components were distributed evenly over the whole cell as visualized by confocal microscopy. Immunohistochemistry of atherosclerotic vessels indicated that PN-1 was found associated with smooth muscle cells, macrophages and platelets. In some regions of the diseased vessels PN-1 was in close proximity to VN and uPAR, but no PN-1 was present in normal vessels. These results indicate a novel function of PN-1 linked to complex formation with uPA that leads to the regulation of VN-dependent adhesion of leukocytes.