Endothelial injury or dysfunction has long been postulated to promote atherogenesis, but structural alterations of endothelium in atherosclerosis have remained obscure. We report the common occurrence of actin-containing stress fibers, stainable by rhodamine-phalloidin, in endothelium overlying atherosclerotic lesions in cholesterol-fed rabbits. Nonlesioned areas in the same aortas showed normal endothelium with minimal development of stress fibers, which was no different from the appearance of endothelium in chow-fed rabbits. Microtubule organization revealed by immunofluorescence appeared normal in all areas. The development of stress fibers may be related to an altered substratum for endothelial attachment. This study provided no evidence to relate stress fiber formation with lesion initiation, but an association with well-developed foam cell lesions was evident.