Glomerular capillary pressure is thought to affect the structure and function of glomerular cells. However, it is unknown whether podocytes are intrinsically sensitive to mechanical forces. In the present study, differentiated mouse podocytes were cultured on flexible silicone membranes. Biaxial cyclic stress (0.5 Hz and 5% linear strain) was applied to the membranes for up to 3 d. Mechanical stress reduced the size of podocyte cell bodies, and processes became thin and elongated. Podocytes did not align in the inhomogeneous force field. Whereas the network of microtubules and that of the intermediate filament vimentin exhibited no major changes, mechanical stress induced a reversible reorganization of the actin cytoskeleton: transversal stress fibers (SF) disappeared and radial SF that were connected to an actin-rich center (ARC) formed. Epithelial and fibroblast cell lines did not exhibit a comparable stress-induced reorganization of the F-actin. Confocal and electron microscopy revealed an ellipsoidal and dense filamentous structure of the ARC. Myosin II, alpha-actinin, and the podocyte-specific protein synaptopodin were present in radial SF, but, opposite to F-actin, they were not enriched in the ARC. The formation of the ARC and of radial SF in response to mechanical stress was inhibited by nonspecific blockade of Ca(2+) influx with Ni(2+) (1 mM), by Rho kinase inhibition with Y-27632 (10 microM), but not by inhibition of stretch-activated cation channels with Gd(3+) (50 microM). In summary, mechanical stress induces a unique reorganization of the actin cytoskeleton in podocytes, featuring radial SF and an ARC, which differ in protein composition. The F-actin reorganization in response to mechanical stress depends on Ca(2+) influx and Rho kinase. The present study provides the first direct evidence that podocytes are mechanosensitive.