The cytoskeletal components of endothelial cells in the renal artery were examined by analysis of en face preparations under confocal laser scanning microscopy. Renal arterial endothelial cells were shown to be elongated along the direction of blood flow, while stress fibers ran perpendicular to the flow in the basal portion. Focal adhesions were observed along the stress fibers in dot-like configurations. On the other hand, stress fibers in the apical portion of cells ran along the direction of flow. The localizations of stress fibers and focal adhesions in endothelial cells in the renal artery differed from those of unperturbed aortic and venous endothelial cells. Tyrosine-phosphorylated proteins were mainly detected at the sites of cell-to-cell apposition, but not in focal adhesions. Pulsatile pressure and fluid shear stress applied over endothelial cells in the renal artery induce stress fiber organization and localization of focal adhesions. These observations suggest that the morphological alignment of endothelial cells along the direction of blood flow and the organization of cytoskeletal components are independently regulated.