Endothelial monolayer integrity is required to maintain endothelial barrier functions and has found to be impaired in several disorders like inflammatory edema, allergic shock or artherosclerosis. Under physiologic conditions in vivo, endothelial cells are exposed to mechanical forces such as hydrostatic pressure, shear stress and cyclic stretch. However, the insight in the effects of hydrostatic pressure on endothelial cell biology is very limited at present. Therefore, in this study we tested the hypothesis that physiologic hydrostatic pressure protects endothelial monolayer integrity in vitro. We investigated the protective efficacy of hydrostatic pressure in microvascular myocardial endothelial (MyEnd) cells and macrovascular pulmonary artery endothelial cells (PAEC) by application of selected pharmacological agents known to alter monolayer integrity in the absence or presence of hydrostatic pressure. In both endothelial cells lines, extracellular Ca²⁺-depletion by EGTA was followed by a loss of VE-cadherin immunostaining at cell junctions. However, hydrostatic pressure (15 cmH₂O) blocked this effect of EGTA. Similarly, cytochalasin D-induced actin depolymerization and intercellular gap formation, cell detachment in response to the Ca²⁺-calmodulin antagonist trifluperazine (TFP), as well as thrombin-induced cell dissociation were also reduced by hydrostatic pressure. Moreover, hydrostatic pressure significantly reduced the loss of VE-cadherin-mediated adhesion in response to EGTA, cytochalasin D and TFP in MyEnd cells as determined by laser tweezer trapping using VE-cadherin-coated microbeads. In caveolin-1-deficient MyEnd cells that lack caveolae, hydrostatic pressure did not protect monolayer integrity compromised by EGTA indicating that caveolae-dependent mechanisms are involved in hydrostatic pressure sensing and signalling.