Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to fluid frictional force (shear stress) induced by blood flow, and shear stress affects the intracellular calcium ([Ca²⁺]_i), which initiates cellular responses. Here, we studied the effect of long-term exposure of shear stress on [Ca²⁺]_i responses in cultured ECs by using a confocal laser microscope and calcium indicator. At the initiation of shear stress of 20 dyn/cm² (0 hr), 27% of the cells exhibited [Ca²⁺]_i responses. This percentage gradually decreased with increasing exposure time, reaching about 4% after 24 hr of exposure. These data indicate that long-term shear-stress exposure affects [Ca²⁺]_i responses in cultured ECs. Furthermore, we studied the effect of magnitude of shear stress on macromolecule uptake. For the low shear-stress, the uptake was enhanced, whereas the uptake was inhibited for higher shear-stress.