The renal glomerulus, the site of plasma ultrafiltration, is exposed to mechanical force in vivo arising from capillary blood pressure and fluid flow. Studies of cultured podocytes demonstrate that they respond to stretch by altering the structure of the actin cytoskeleton, but the mechanisms by which physical force triggers this architectural change and the signaling pathways that lead to generation of second messengers are not defined. In the present study, we found that in renal epithelial cells (podocytes and MDCK cells), application of mechanical force to the cell surface through fibronectin-coated ferric beads and exposure of the cells to magnetic force, leads to Rho translocation and actin cytoskeleton reorganization. This applica tion of force recruited Rho and filamentous actin (F-actin) to bead loci and subsequently stimulated phospholipase D (PLD), a downstream effector of Rho. Using MDCK cells that stably express regulators of G protein signaling (RGS) proteins (RGS4 attenuates G_i and G_q, and the p115RhoGEF-RGS domain [p115-RGS] attenuates G_12/13) to define the signaling pathway, we found that mechanical force induced G_12/13-Rho activation and increased F-actin to stimulate PLD activity. The activation can be partially prevented by C₃ exoenzyme. Pretreatment of the cells with chemical inhibitors of several kinases showed that calmodulin-dependent kinase is also involved in stretch-induced PLD activation by a separate pathway. Taken together, our data demonstrate that in cultured podocytes and MDCK cells, mechanical force leads to actin cytoskeleton reorganization and PLD activation. The signaling pathways for PLD activation involve G_12/13/Rho/F-actin and calmodulin-dependent kinase.