Hemodynamic forces in the form of shear stress (SS) and mechanical strain imposed by circulating blood are recognized factors involved in the control of systemic endothelial cell (EC) cytoskeletal structure and function. However, the effects of acute SS on pulmonary endothelium have not been precisely characterized, nor the mechanism of rapid SS-induced EC cytoskeletal rearrangement understood. We exposed bovine and human pulmonary EC monolayers to laminar SS (10 dynes/cm2) in a parallel plate flow chamber and observed increased actin stress fiber formation 15 min after application of flow. Acute SS-induced pronounced cortical cytoskeletal rearrangement characterized by myosin light chain kinase (MLCK)- and Rho-associated kinase (RhoK)-dependent accumulation of diphosphorylated regulatory myosin light chains (MLC) in the cortical actin ring, junctional protein tyrosine phosphorylation, and transient peripheral translocation of cortactin, an actin-binding protein involved in the regulation of actin polymerization. SS-induced cortactin translocation was independent of Erk-1,2 MAP kinase, p60(Src), MLCK, or RhoK activities, and unaffected by overexpression of a cortactin mutant lacking four major p60(Src) phosphorylation sites. However, both SS-induced transient cortactin translocation and cytoskeletal reorientation in response to sustained (24 h) SS was abolished in cells overexpressing either dominant negative Rac 1 or a dominant negative construct of its downstream target, p21-activated kinase (PAK)-1. Our results suggest a potential role for cortactin in the SS-induced EC cortical cytoskeletal remodeling and demonstrate a novel mechanism of Rac GTPase-dependent regulation of the pulmonary endothelial cytoskeleton by SS.