Blood flow patterns play an important role in cardiovascular development, as changes can cause congenital heart malformations. Shear stress is positively correlated to blood flow. Therefore, it is likely that shear stress is also involved in cardiac development. In this study, we investigated the expression patterns of ET‐1, NOS‐3, and KLF‐2 mRNA in a series of developmental stages of the chicken embryo. These genes are reported to be shear responsive. It has been demonstrated that KLF‐2 is confined to areas of high shear stress in the adult human aorta. From in vitro studies, it is known that ET‐1 is down‐regulated by shear stress, whereas NOS‐3 is up‐regulated. Therefore, we expect ET‐1 to be low or absent and NOS‐3 to be high at sites where KLF‐2 expression is high. Our study shows that, in the early stages, expression patterns are mostly not shear stress‐related, whereas during development, this correlation becomes stronger. We demonstrate overlapping expression patterns of KLF‐2 and NOS‐3 in the narrow parts of the cardiovascular system, like the cardiac inflow tract, the atrioventricular canal, outflow tract, and in the early stages in the aortic sac and the pharyngeal arch arteries. In these regions, the expression patterns of KLF‐2 and NOS‐3 exclude that of ET‐1. Our results suggest that, in the embryonic cardiovascular system, KLF‐2 is expressed in regions of highest shear stress, and that ET‐1 and NOS‐3 expression, at least in the later stages, is related to shear stress. Developmental Dynamics 230:57–68, 2004. © 2004 Wiley‐Liss, Inc.