F ocal adhesion sites were observed in cultured endothelial cells by tandem scanning confocal microscopy and digitized image analysis, techniques that provide real-time images of adhesion site area and topography in living cells. Image subtraction demonstrated that in the presence of unidirectional steady laminar flow (shear stress [tau] = 10 dyn/cm2) a substantial fraction of focal adhesion sites remodeled in the direction of flow. In contrast, focal adhesions of control (no flow) cells remodeled without preferred direction. In confluent monolayers subjected to shear stresses of 10 dyn/cm2, cells began to realign in the direction of flow after 7-9 h. This was accompanied by redistribution of intracellular stress fibers, alignment of individual focal adhesion sites, and the coalescence of smaller sites resulting in fewer, but larger, focal adhesions per cell. Cell adhesion, repeatedly calculated in the same cells as a function of the areas of focal contact and the separation distances between membrane and substratum, varied by < 10% during both short (30 min), or prolonged (< or = 24 h), periods of exposure to flow. Consistent with these measurements, the gains and losses of focal adhesion area as each site remodeled were approximately equivalent. When the glass substratum was coated with gelatin, rates of remodeling were inhibited by 47% during flow (tau = 10 dyn/cm2). These studies: (a) reveal the dynamic nature of focal adhesion; (b) demonstrate that these sites at the ablumenal endothelial membrane are both acutely and chronically responsive to frictional shear stress forces applied to the opposite (lumenal) cell surface; and (c) suggest that components of the focal adhesion complex may be mechanically responsive elements coupled to the cytoskeleton.