Adaptation of the endothelial cytoskeleton to an altered hemodynamic environment has been detected using indirect fluorescence labeling in fixed cells, but acute changes in cytoskeletal movement may play a role in rapid flow-mediated signaling by redistribution of forces to remote sites in the cell. For the first time, access to the dynamics of the cytoskeleton in living cells is possible by using chimeric constructs of green fluorescent protein (GFP). Endothelial cells expressing GFP-vimentin were exposed to unidirectional steady laminar shear stress in a parallel plate flow chamber. Deconvolution of fluorescence optical sections generated a precise 3-D spatial description of the corresponding intermediate filament (IF) network. IF motion was rapidly altered in regions of the cell after a step change in shear stress applied to the lumenal surface. A bioengineering analysis of real time cytoskeletal dynamics in living cells suggests a complex integration of biomechanics with spatial organization of intracellular signaling.