Stress fiber expression and fibronectin distribution were studied in perfusion-fixed,en facepreparations of chick aorta and mesenteric artery stained with fluorescent phalloidin, anti-fibronectin or anti-vinculin. Transmission electron microscopy, ultrastructural immuno-cytochemistry and confocal laser scanning microscopy were also used. Thick stress fibers aligned parallel to the vessel axis were prevalent in the aorta and the proximal part of the mesenteric artery, but their number and caliber decreased with vessel size. Fibronectin fibrils were also aligned parallel to the direction of blood flow in the aorta and the proximal portion of the mesenteric artery, the degree of alignment gradually decreasing as the mesenteric artery diminished in size. Stress fibers and fibronectin fibrils were often colinear and appeared to be tightly coupled, forming a structure similar to the adhesion plaque of cultured cells. In a small area immediately downstream from a flow divider, endothelial cells were polygonal and both stress fiber expression and axial fibronectin organization were lost. Irregular fibronectin patterns were also seen in the distal part of the mesenteric artery, which contained aligned endothelial cells with only a few or no stress fibers. No correlation between cell shape and fibronectin patterns was noted. Endothelial cell stress fibers, whose expression is influenced by fluid flow, may have a role in the alignment of fibronectin in large arteries.