The luminal surface of rat lung microvascular endothelial cells in situ is sensitive to changing hemodynamic parameters. Acute mechanosignaling events initiated in response to flow changes in perfused lung microvessels are localized within specialized invaginated microdomains called caveolae. Here we report that chronic exposure to shear stress alters caveolin expression and distribution, increases caveolae density, and leads to enhanced mechanosensitivity to subsequent changes in hemodynamic forces within cultured endothelial cells. Flow-preconditioned cells expressed a fivefold increase in caveolin (and other caveolar-residing proteins) at the luminal surface compared with no-flow controls. The density of morphologically identifiable caveolae was enhanced sixfold at the luminal cell surface of flow-conditioned cells. Laminar shear stress applied to static endothelial cultures (flow step of 5 dyn/cm2), enhanced the tyrosine phosphorylation of luminal surface proteins by 1.7-fold, including caveolin-1 by 1.3-fold, increased Ser1179 phosphorylation of endothelial nitric oxide synthase (eNOS) by 2.6-fold, and induced a 1.4-fold activation of mitogen-activated protein kinases (ERK1/2) over no-flow controls. The same shear step applied to endothelial cells preconditioned under 10 dyn/cm2 of laminar shear stress for 6 h and induced a sevenfold increase of total phosphotyrosine signal at the luminal endothelial cell surface enhanced caveolin-1 tyrosine phosphorylation 5.8-fold and eNOS phosphorylation by 3.3-fold over static control values. In addition, phosphorylated caveolin-1 and eNOS proteins were preferentially localized to caveolar microdomains. In contrast, ERK1/2 activation was not detected in conditioned cells after acute shear challenge. These data suggest that cultured endothelial cells respond to a sustained flow environment by directing caveolae to the cell surface where they serve to mediate, at least in part, mechanotransduction responses.