Smooth muscle cell (SMC) rarefaction is involved in the development of several vascular pathologies. We suggest that the plasminogen activation system is a potential extracellular signal that can induce pericellular proteolysis and apoptosis of vascular SMCs. Using primary cultures of arterial SMCs, we show that plasmin generated from plasminogen on the cell surface induces cell retraction and fibronectin fragmentation, leading to detachment and morphological/biochemical changes characteristic of apoptosis (also called anoikis). The generation of cell-bound plasmin mediated by tissue-type plasminogen activator (t-PA), constitutively expressed by VSMCs, requires binding of plasminogen to the cell surface and is inhibited by epsilon-aminocaproic acid (IC50=0.9+/-0.2 mM), a competitor of plasminogen binding to membrane glycoproteins. Conversely, addition of alpha2-antiplasmin, which blocks free plasmin in the cell supernatant, could not fully prevent anoikis. Finally, an MMP inhibitor failed to prevent VSMC anoikis, arguing for a direct involvement of plasmin in this phenomenon. Indeed, similar changes are induced by plasmin directly added to VSMCs or to arterial rings, ex-vivo. We show for the first time that pathological anoikis can be triggered by a process that requires functional assembly of the plasminogen activation system on the surface of VSMCs.