The available data on F-actin microfilament distribution in vascular endothelial cells in vivo is limited. In this study, the appearance and distribution of endothelial cell microfilaments in the rabbit thoracic aorta, the abdominal aorta and its major arterial branch points, and the aortic bifurcation were examined. Perfusion fixed rabbit aortas were stained in situ for F-actin by infusing rkodamine phalloidin via a peristaltic pump into the aortas at a slow flow rate. This new technique resulted in excellent visualization of branch points and allowed for a precise description of the actin microfilament bundles in endothelial cells along flow dividers. In the thorack and abdominal aorta, away from branch ostia, actin microfilaments were localized in two regions of the endothelial cells, as a prominent band that completely outlined the cell periphery, and also as short central stress fibers. The central stress fibers were more frequent and prominent in cells of the abdominal aorta. At branch sites and at the aortic bifurcation, long, thick microfilament bundles were present in endothelial cells extending from the tip of the flow divider to a few millimeters along the branch arteries, the aorta, and the iliac arteries. Peripheral actin, however, no longer completely surrounded the cells. The thick bundles were not prominent in endothelial cells located adjacent to the proximal Up of branches or at the iliac arteries opposite the flow divider. This study shows that endothelial cell F-actin microfilament distribution in vivo is well defined along the aortic-arterial system. The prominent central microfilament bundles and the reduced peripheral microfilaments seen at localized regions may reflect an adaptive response to elevated shear stress at these sites.