It has been proposed previously that actin filaments and cell adhesion sites are involved in mechanosignal transduction. In this study, we present certain morphological evidence that supports this hypothesis. The 3D disposition of actin filaments and phosphotyrosine-containing proteins in endothelial cells in situ was analyzed by using confocal microscopy and image reconstruction techniques. Surgical coarctations were made in guinea pig aortas, and the same 3D studies were conducted on such areas 1 week later. Stress fibers (SFs) were present at both basal and apical regions of endothelial cells regardless of coarctation, and several phosphotyrosine-containing proteins were associated with SF ends. Apical SFs had one end attached to the apical cell membrane and the other attached to either the basal membrane or the lateral cell border. Within the coarctation area, the actin filament-containing and vinculin-containing structures became prominent, especially at the apical and the lateral regions. Substantially higher levels of anti-phosphotyrosine and anti-Src staining were detected in the constricted area, particularly at the cell-cell apposition, whereas the anti-focal adhesion kinase, anti-CT10-related kinase, anti-platelet endothelial cell adhesion molecule-l, anti-vinculin, and phalloidin staining intensities increased only slightly after coarctation. We propose that apical SFs directly transmit the mechanical force of flow from the cell apex to the lateral and/or basal SF anchoring sites and that the SF ends associated with signaling molecules are sites of signal transduction. Our results support the idea that the cell apposition area is the major fluid shear stress-dependent mechanosignal transduction site in endothelial cells.