The activation of Rho-kinase is known to modulate the organization of the actin-based cytoskeletal systems, including the formation of stress fibers and focal adhesions. Rho-kinase likely plays a more crucial and complex role in the organization of actin-based cytoskeletal systems than in that of myosin light chain kinase (MLCK). In order to understand the role of Rho-kinase in the organization of stress fibers and focal adhesions, we treated cultured fibroblasts with a Rho-kinase inhibitor and analyzed the stress fiber and focal adhesion organization under conventional fluorescence microscopy and replica electron microscopy. Some of the cells were transfected with GFP-labeled paxillin, actin or alpha-actinin, and the effects of the inhibitor were monitored in the living cells. The Rho-kinase inhibitor caused disassembly of the stress fibers and focal adhesions in the central portion of the cell within 1 h. However, the stress fibers and focal adhesions located in the cell periphery were not as severely affected by the Rho-kinase inhibitor. The time-lapse video recording revealed that when these cells were washed with a fresh medium in order to remove the Rho-kinase inhibitor, the stress fibers and focal adhesions located in the center of the cell gradually reorganized and, within 1.5-2 h, the cells completely recovered. This observation strongly suggests that the activation of Rho-kinase plays an important role in the organization of the central stress fibers and focal adhesions.