Endothelial cells respond to mechanical stresses of the circulation with cytoskeletal rearrangements such as F-actin stress fiber alignment along the axis of fluid flow. Endothelial cells are also exposed to hypertonic stress in the renal medulla or during mannitol treatment of cerebral edema. We report here that arterial endothelial cells exposed to hypertonic stress rearrange F-actin into novel actin-myosin II fibers with regular 0.5 µm striations, in which -actinin colocalizes with actin. These striated fibers assemble over hours into 3-dimensional, irregular, polygonal actin networks most prominent at the cell base, occasionally surrounding the nucleus with a geodesic-like structure. Hypertonicity-induced assembly of striated polygonal actin networks was inhibited by cytochalasin D, blebbistatin, cell ATP depletion, and intracellular Ca²⁺ chelation, but did not require intact microtubules, regulatory volume increase, or de novo synthesis of RNA or protein. Striated polygonal actin network assembly was insensitive to inhibitors of MAP kinases, tyrosine kinases, or PI3-kinase, but was prevented by C3 exotoxin, by the ROCK inhibitor Y-27632, and by overexpressed dominant negative RhoA. In contrast, overexpression of dominant negative Rac or of dominant negative cdc42 did not prevent striated polygonal actin network assembly. The actin networks described here are novel in form, as striated actin-myosin structures of non-muscle cells, as a cellular response to hypertonicity, and as a cytoskeletal regulatory function of RhoA. Endothelial cells may use RhoA-dependent striated polygonal actin networks, possibly in concert with cytoskeletal load-bearing elements, as a contractile, tension-generating component of their defense against isotropic compressive forces.