Cell-membrane fluidity modulates protein function and mobility. Blood flow-associated shear stress induces changes in endothelial cell functions, many of which may be initiated by alterations in the plasma membrane. In this study, we quantify the effects of shear stress on endothelial cell-membrane lipid-fluidity measured by fluorescence recovery after photobleaching (FRAP). A confocal microscope was used for FRAP-measurements on DiI-stained bovine aortic endothelial cells in a parallel-plate flow chamber under static conditions, after a step-shear stress of 10 dynes/cm2 which was maintained for 15 minutes, and after the removal of shear stress. The DiI-diffusion coefficient (D) increased immediately and significantly from a pre-step value of 4.3±0.48×10-9 to 8.7±1.8×10-9 cm2/sec within 2 min after the initiation of shear stress and remained elevated thereafter. D returned to control values within 1 minute after cessation of shear stress. The novel measurements of shear-induced increases in membrane-fluidity provide important insight into the mechanisms of shear-induced protein modulation and mobilization.