Endothelial cells line the vasculature and are exposed to mechanical shear stress because of blood motion. Previous studies have shown that endothelial cells respond to shear stress by altering their metabolism and genetic expression, but the mechanism for shear stress signal transduction remains unclear. In the current study, we investigated the role of intracellular Ca²⁺ increases as a part of the shear stress signal transduction cascade. Primary human umbilical vein endothelial cells were loaded with the calcium-sensitive dye fura-2 and exposed to fluid flow in a parallel plate flow chamber in the presence of the inflammatory mediator histamine or the proteolytic enzyme thrombin. The initiation of shear stress (in the range of 0.2-20 dyne/cm²) in the absence of either agonist caused no increase in intracellular Ca²⁺ levels. Cells exposed to either histamine (10^–9 to 10^–7 mol/l) or thrombin (0.02-0.2 u/ml) showed an intracellular calcium increase (20-150 nmol/L) that was dependent on the magnitude of the shear stress and on the concentration of agonist. In cells exposed to histamine and shear stress, the magnitude of the intracellular calcium increase was not altered, except at 10^–7 mol/L histamine. The time course of the response was significantly faster for arterial than for venous levels of shear stress at histamine concentrations from 10^–9 to 10^–7 mol/L. The magnitude of the [Ca²⁺] response was dependent on both the magnitude of the shear stress and the concentration of thrombin. At a thrombin concentration of 0.2 U/mL, the increase in intracellular Ca²⁺ was significantly greater at arterial levels of shear stress (6-20 dyne/cm²) than at venous levels of shear stress (0.2-1 dyne/cm²). Because we solved the governing mass balance equation to precisely determine the effect of flow on local agonist concentration, the alterations in the [Ca²⁺] response were not due to differences in mass transfer characteristics. These results demonstrate that even in a system in which the initiation of shear stress without agonist causes no detectable change in intracellular Ca²⁺, the calcium response to agonists is changed, which suggests that the signal transduction pathway for shear stress acts synergistically with the thrombin and histamine signal transduction pathways. (J Vasc Surg 2000;32:593-601.)