In vivo and ex vivo studies with whole vessels demonstrated that the endothelium comprises the major permeability barrier in the blood vessel wall. As a lining between the blood and the underlying tissue, the endothelium is exposed to biochemical components of the blood and wall shear stress. The endothelium responds to these biochemical and mechanical agonists both metabolically and morphologically. Using a model system consisting of human umbilical vein endothelial cells cultured on a permeable polycarbonate membrane, the permeability barrier function of endothelial monolayers was characterized quantitatively by determination of permeability coefficients. The effect of biochemical agonists and wall shear stress on permeability was assessed by the extent to which the agonists modulated the baseline permeability coefficient. Permeability increased nearly 10-fold over baseline values in the presence of 0.1 U/ml thrombin. Using altered forms of thrombin, it was shown that both proteolytic activity and binding to the endothelium are necessary for thrombin to increase permeability. Permeability was unaltered in the presence of 1 μM bradykinin. Permeability decreased to 25% of baseline during treatment with 6 nM iloprost, a stable functional analog of the vasodilator and anti-platelet aggregating agent prostacyclin. Prostacyclin often acts through the second messenger cAMP. The permeability also decreased in cells treated with combinations of cAMP analogs and phosphodiesterase inhibitors which act in concert to increase intracellular cAMP concentrations. Another anti-platelet aggregating agent and vasodilator, endothelial derived relaxing factor, acts by increasing intracellular cGMP concentrations. The cGMP analog dibutyryl cGMP also decreased endothelial permeability. Pretreatment of endothelial monolayers with iloprost, cAMP analog/phosphodiesterase inhibitor combinations or dibutyryl cGMP diminished the permeability increasing effect of thrombin. A parallel plate flow chamber was used to subject endothelial monolayers cultured on permeable membranes to steady, laminar flow under the condition of no volume flux across the membrane. After 1 hour of 7 dynes/cm\sp2 shear stress, permeability increased 7-fold in a partially reversible manner. When cells were pretreated with 6 nM iloprost, the effect of shear stress was diminished to a 2-fold increase which was completely reversible.