Endothelial cells (ECs) respond to fluid shear stress. They reveal shear stress related morphological changes in both their cell shape and cytoskeletal organization. Little is known about the cytoskeletal organization of ECs in situ. We studied, together with the living ultrasound high resolution imaging system, the distribution of stress fibers (SFs), certain focal adhesion (FA) and signal transduction associated proteins in guinea pig aortic and venous ECs. Although SFs present in the basal portion of venous ECs ran along the direction of the blood flow, their size was smaller and their number was fewer than those of aortic ECs. Venous ECs were elongated to the direction of flow than in aortic ECs exposed over normal shear stress (SS). Since fluid SS in the vein is low, a sustained and uni-directional low SS over a long period might thus cause these structural features observed in venous ECs.