Locomoting cells exhibit a polarity whereby certain organelles, like the centrosome, and cytoskeletal structures, like stress fibers, are preferentially oriented in the direction of migration. To determine if this was also true in endothelial cells (ECs) of the rat aorta that are migrating toward the heart, whole mounts of abdominal and thoracic aorta were double stained with rhodamine phalloidin to label stress fibers and sera that labels centrosomes. Our results show that in 66% of the ECs of the abdominal aorta where stress fibers were present, 47% had stress fibers on the heart side of the nucleus, 21% had stress fibers on the side of the nucleus away from the heart, and 32% had stress fibers across the cell. Similarly, in 50% of the ECs of the thoracic aorta where stress fibers were present, these values were 56, 19, and 25%, respectively. The results also showed that the centrosome was preferentially located toward the heart in the majority (61%) of the ECs with stress fibers as well as in ECs without stress fibers. Since in both, the same percentage of ECs had centrosomes preferentially oriented toward the heart, these results imply that while the centrosome may determine the position of the stress fibers, the stress fibers do not appear to determine the position of the centrosome. Nevertheless, both centrosomes and stress fibers in aortic ECs are preferentially oriented in the direction of migration, where they may be involved in defining the direction and providing the force for locomotion, respectively.