Vascular remodelling can often be observed as consequence of chronic changes in blood flow. Whereas arteriogenesis is thought to be induced by elevated flow, a decrease in flow may result in plaque formation or atherosclerosis. Both processes are tightly controlled by proteases and their individual inhibitors. Podosomes are actin-rich cell protrusions which are locally associated with enhanced proteolysis of the extracellular matrix. To delineate the role of podosomes for endothelial matrix degradation, we studied the gelatinolytic activity and cellular arrangement of podosomes in endothelial cells exposed to shear stress. Human umbilical vein endothelial cells (HUVEC, passage 2 to 3) were grown on fluorescent gelatin (Oregon green-conjugated) whose structure was imaged by confocal microscopy. The cells constitutively formed podosomes that were identified via costaining of cortactin and F-actin. Gelatinolytic activity of HUVECs was quantified according to gelatin fluorescence and to the density of circular gelatin defects. Shear stress was produced by unidirectional perfusion of a parallel plate flow chamber (Ibidi). Cells grown under static conditions expressed podosomes either in even distribution or in circular arrangements (rosettes) that were associated with corresponding structures of gelatin degradation. Rosette density and gelatin degradation were highly increased after 2 hours of protein kinase C (PKC) stimulation by PMA (phorbol-12-myristate-13-acetate, 50 ng/ml) (reduction of gelatin fluorescence to 68% of control). Inhibition of matrix metalloproteinase (MMP) activity by GM6001 (15 µM) virtually abolished the occurrence of gelatin defects in untreated and PMA-treated endothelial cells. Exposure to shear stress for 48 hours altered the density of ring-shaped gelatin defects of 5 – 30 µm in diameter. Under very low shear stress (< 1 dyn/cm2) the amount of rosette imprints was significantly increased compared to physiological flow (10 – 20 dyn/cm2) and medium flow conditions (2 – 4 dyn/cm2) (high flow 45 ± 10, medium flow 43 ± 28 vs. low flow 83 ± 7 per mm2, n = 3, means ± S.D., ANOVA). HUVECS demonstrated typical alignment under high shear stress, but no signs of altered cell density or damage. Thus gelatinolytic activity of endothelial cells is locally upregulated at podosome rosettes under low flow conditions, as compared to physiological arterial shear stress. This effect is presumably produced by an increased number in podosome rosettes recruiting higher MMP activities. Since matrix degradation promotes cell migration and impairs vessel stability, shear stress is critically important to stabilize the structure of the vascular wall under physiological conditions through mechanisms that locally regulate proteolytic activities.