In this study, we investigated heat shock protein (HSP) expression and stress fiber (SF) formation in endothelial cells (ECs) within the arterial vascular tree of adult rats under normal physiological conditions. Using quantitative immunofluorescence microscopy, we found no significant differences in expression of HSPs 25, 60, 70, and 90 among ECs in the straight portions of rat arteries. In these regions, ECs appeared spindle-shaped and contained short bundles of central SFs. In contrast, ECs in the curved portions or the branch sites of the arteries, exhibited striking differences in HSP expression. ECs with higher HSP expression were localized at the lesser curvature in the curved portions or the distal site of the branch ostia. Moreover, the ECs became polygonal and contained irregular central SFs at the lesser curvature. At the branch sites, downstream ECs became spear-shaped and contained long, thick bundles of central SFs. Curved portions or branch sites are the regions of disturbed flow at which early atherosclerotic lesions are often found. Our results demonstrate these positional differences in HSP expression associated with changes in SF formation within the arterial vascular tree under non-pathological conditions. Our study provides basic information for understanding stress responses via HSP expression and SF formation in vascular ECs and the pathogenesis of atherosclerotic disease.