Shear stress protects endothelium from a variety of risk factors for vascular disease. Here, we demonstrate a novel mechanism whereby shear stress inhibited reactive oxygen species (ROS)-triggered signaling cascades in endothelial cells. Stimulation of bovine aortic endothelial cells (BAECs) with H(2)O(2) induced a 3.07-fold increase in p66(Shc) phosphorylation. This response was fully blocked by pretreatment of cells with specific JNK but not p38 or ERK MAP kinase inhibitor. Further study showed that knocking down of apoptosis signal-regulating kinase 1 (ASK1) by siRNA transfection in cells dramatically inhibited phosphorylation of JNK and p66(Shc) elicited by H(2)O(2). Pre-perfusion of BAECs cultured in silastic tubes with laminar flow generated by a servo-pump system for 30 min also significantly suppressed H(2)O(2)-induced phosphorylation of p66(Shc). This was accompanied by quantitatively similar inhibition of ASK1 and JNK phosphorylation and activation. These results suggested that shear stress protects endothelium against oxidant stress by suppression of ASK1-JNK-mediated p66(Shc) phosphorylation.