The mechanical properties of cultured bovine aortic endothelial cells exposed to a fluid-imposed shear stress were studied using the micropipette technique. The cells, which were attached to a Thermanox plastic substrate, were exposed to a specific steady shear stress of either 10, 30, or 85 dynes/cm2 and for a duration ranging from 0.5 to 24 hours. Morphological changes in shape and orientation were observed, and following each experiment, the mechanical properties were measured using the micropipette aspiration technique applied to cells detached from the substrate. Fluorescent microscopy was carried out to observe cytoskeletal F-actin filaments stained with rhodamine phalloidin. During exposure to shear, the en face shape of the endothelial cells on the substrate became more elongated and their long axis became oriented to the direction of flow. There was also an alteration in the F-actin filaments. These changes were dependent on both the level of shear stress and the duration of exposure. After detachment, the cells exposed to shear maintained their deformed shape. This is in contrast to cells in a static, no-flow environment which became spherical in shape upon detachment. Cells exposed to shear stress demonstrated a mechanical stiffness significantly greater than that of control cells, which was dependent on both the level of shear stress and the duration of exposure. Furthermore, it appears that the influence of shear stress on endothelial cell mechanical stiffness may be related to alterations in cytoskeletal structure.