Podocytes are exposed to mechanical forces arising from glomerular capillary pressure and filtration. It has been shown that stretch affects podocyte biology in vitro and plays a significant role in the development of glomerulosclerosis in vivo. However, whether podocytes are sensitive to fluid shear stress is completely unknown. In the present study, we therefore exposed cells of a recently generated conditionally immortalized mouse podocyte cell line to defined fluid shear stress in a flow chamber, mimicking flow of the glomerular ultrafiltrate over the surface of podocytes in Bowman's space. Shear stress above 0.25 dyne/cm² resulted in dramatic loss of podocytes but not of proximal tubular epithelial cells (LLC-PK1 cells) after 20 h. At 0.015-0.25 dyne/cm², lamellipodia formation in podocytes was enhanced and the actin nucleation protein cortactin was redistributed to the cell margins. Shear stress further diminished stress fibers and the presence of vinculin in focal adhesions. Linear ZO-1 distribution at cell-cell contacts remained unaffected at low shear stress. At 0.25 dyne/cm² the monolayer was broken up and remaining cell-cell contacts were reinforced by F-actin and -actinin. Since the cytoskeletal changes induced by shear stress suggested the involvement of tyrosine kinases (TKs), we tested several TK inhibitors which were all without effect on podocyte number under static conditions. At 0.25 dyne/cm², however, the TK inhibitors genistein and AG 82 were associated with marked podocyte loss. Our data demonstrate that podocytes are highly sensitive to fluid shear stress. Shear stress induces reorganization of the actin cytoskeleton and activates specific tyrosine kinases.