Flow-related shear stress has been shown to modulate endothelial cell structure and function including F-actin microfilament organization. Focal adhesion-associated proteins such as vinculin, talin, and specific integrins may play a role in the modulation of these cytoskeletal and morphological changes. Double-label immunofluorescence studies indicated that, in static culture, α5β1 fibronectin receptors (α5β1 FNRs) and αvβ3 vitronectin receptors (αvβ3 VNRs) were found predominantly in the peripheral regions of bovine aortic endothelial cells (BAECs) corresponding to the localization of vinculin, talin, and actin microfilament terminations. In response to shear stress, concomitant with cell elongation and the appearance of stress fibers aligned with the direction of flow, there was a prominent localization of vinculin and αvβ3 VNRs as the “upstream” end of the cells. Stress fiber terminations were clearly evident at these concentrations of focal adhesion-associated proteins. These data suggest that the upstream concentration of these proteins may direct shear stress-induced stress fiber formation and may function in the alignment of the fibers in the direction of flow. Levels of surface αvβ3 VNRs were found to decrease in response to flow, possibly reflecting the decrease in numbers of “downstream” receptors. Unlike the arrangement of vinculin and αvβ3 VNRs observed following exposure to flow, talin and α5β1 FNRs, in addition to being localized at the upstream end of the cell, were also evenly distributed throughout the rest of the cell. Surface levels of α5β1 FNRs increased in response to shear stress, perhaps providing an increased adherence of BAECs to the extracellular matrix through these receptors. These data suggest that focal adhesion-associated proteins play specific roles in the response of BAECs to shear stress. © 1995 Wiley-Liss, Inc.