Fluid shear stress greatly influences the biology of vascular endothelial cells and the pathogenesis of atherosclerosis. Endothelial cells undergo profound shape change and reorientation in response to physiological levels of fluid shear stress. These morphological changes influence cell function; however, the processes that produce them are poorly understood. We have examined how actin assembly is related to shear-induced endothelial cell shape change. To do so, we imposed physiological levels of shear stress on cultured endothelium for up to 96 hours and then permeabilized the cells and exposed them briefly to fluorescently labeled monomeric actin at various time points to assess actin assembly. Alternatively, monomeric actin was microinjected into cells to allow continuous monitoring of actin distribution. Actin assembly occurred primarily at the ends of stress fibers, which simultaneously reoriented to the shear axis, frequently fused with neighboring stress fibers, and ultimately drove the poles of the cells in the upstream and/or downstream directions. Actin polymerization occurred where stress fibers inserted into focal adhesion complexes, but usually only at one end of the stress fiber. Neither the upstream nor downstream focal adhesion complex was preferred. Changes in actin organization were accompanied by translocation and remodeling of cell-substrate adhesion complexes and transient formation of punctate cell-cell adherens junctions. These findings indicate that stress fiber assembly and realignment provide a novel mode by which cell morphology is altered by mechanical signals.