In response to externally applied shear stress, cultured endothelial monolayers develop prominent, axially-aligned, microfilamentous bundles, termed "stress fibers" (Dewey: Journal of Biomechanical Engineering 106:31-35, 1984; Franke et al.: Nature 81:570-580, 1984; Franke et al.: Klin. Wochenschr 64:989-992, 1986; Wechezak et al.: Laboratory Investigation 53:639-647, 1985). It is unclear, however, whether similar stress fibers develop in noncontiguous endothelial cells and whether these structures are necessary for adherence of individual cells under shear stress. It also is unknown what alterations occur in microtubules, intermediate filaments, and focal contacts as a consequence of shear stress. In this study, endothelial cells, free of intercellular contact, were exposed to 93 dynes/cm2 for 2 hr. With the aid of specific labeling probes and interference reflection microscopy, the distributional patterns of microfilaments, microtubules, intermediate filaments, and focal contacts were examined. Following shear stress, microfilament bundles and their associated focal contacts were concentrated in the proximal (relative to flow direction) cell regions. In contrast, microtubules were distributed uniformly within cell contours. Intermediate filaments displayed only an occasional tendency for accumulation at proximal edges. When cells were shear-tested in the presence of cytochalasin B to inhibit microfilament assembly, considerable cell loss occurred. Following inhibition of tubulin polymerization, no increase was observed in the percentage of cells lost due to shear over nontreated controls. Nocodazole-treated cells, however, were characterized by prominent stress fibers throughout the cell. These results indicate that stress fiber and focal contact reorganization represent major responses in isolated endothelial cells exposed to shear stress and that these cytoskeletal structures are necessary for adherence.