Hemodynamic shear stress affects endothelial cell structure and function, but little is known about the signal transduction mechanisms involved in these processes. The effect of laminar shear stress on cytosolic pH (pHi) was examined in rat aortic endothelial cells cultured in glass capillary tubes. Shear stress forces led to a rapid decrease in pHi (maximal effect 0.09 pH unit at 13.4 dynes per square centimeter). Removal of specific ions or addition of exchange inhibitors suggests that in vascular endothelial cells shear stress forces activate both an alkali extruder, sodium ion-independent chloride-bicarbonate ion exchange, and an acid extruder, sodium-hydrogen ion exchange; the net effect in physiologic buffer with the bicarbonate ion is a decrease in pHi.