Endothelial hyper- permeability leading to vascular leak is an important consequence of sepsis and sepsis-induced lung injury. We previously reported that heat shock protein (hsp) 90 inhibitor pretreatment improved pulmonary barrier dysfunction in a murine model of sepsis-induced lung injury. We now examine the effects of hsp90 inhibitors on LPS-mediated endothelial hyper-permeability, as reflected in changes in transendothelial electrical resistance (TER) of bovine pulmonary arterial endothelial cells. Vehicle-pretreated cells exposed to endotoxin exhibited a concentration-dependent decrease in TER, activation of pp60^Src , phosphorylation of the focal adhesion protein paxillin and reduced expression of the adherens junction (AJ) proteins, VE-cadherin and -catenin. Pretreatment with the hsp90 inhibitor, radicicol, prevented the decrease in TER, maintained VE-cadherin and -catenin expression, inhibited activation of pp60^Src and phosphorylation of paxillin. Similarly, when BPAEC hyper-permeability was induced by endotoxin-activated neutrophils, pretreatment of neutrophils and/or endothelial cells with radicicol protected against the activated neutrophil-induced decrease in TER. Increased paxillin phosphorylation and decreased expression of -catenin and VE-cadherin were also observed in mouse lungs, 12hr after endotoxin (ip) and were attenuated in mice pretreated with radicicol. These results suggest that hsp90 plays an important role in sepsis-associated endothelial barrier dysfunction.