Technical Abstract: Bacterial lipopolysaccharide (LPS) is a key mediator in the vascular leak syndromes associated with Gram-negative bacterial infections. LPS opens the paracellular pathway in pulmonary vascular endothelia through protein tyrosine phosphorylation. We now have identified the protein tyrosine kinase (PTK)s and their substrate(s) required for LPS-induced protein tyrosine phosphorylation and opening of the paracellular pathway in human lung microvascular endothelial cell (HMVEC-L)s. LPS disrupted barrier integrity in a dose- and time-dependent manner and prior broad-spectrum PTK inhibition was protective. LPS increased tyrosine phosphorylation of zonula adherens (ZA) proteins, VE cadherin, gamma-catenin and p120ctn. Two src family PTK (SFK)-selective inhibitors, PP2 and SU6656, blocked LPS-induced increments in tyrosine phosphorylation of VE cadherin and p120ctn and paracellular permeability. In HMVEC-Ls, c-src, yes, fyn, and lyn were expressed at both mRNA and protein levels. Selective siRNA-induced knockdown of c-src, fyn or yes diminished LPS-induced src Y416 phosphorylation, tyrosine phosphorylation of VE-cadherin and p120ctn, and barrier disruption whereas knockdown of lyn had no such effects. For VE-cadherin phosphorylation, knockdown of either c-src or fyn provided total protection whereas yes knockdown was only partially protective. For p120ctn phosphorylation, knockdown of fyn, c-src, or yes each provided comparable but partial protection. Toll-like receptor (TLR)4 was expressed both on the surface and intracellular compartment of HMVEC-Ls. Prior knockdown of TLR4 blocked both LPS-induced SFK activation and barrier disruption. These data indicate that LPS recognition by TLR4 activates c-src, fyn and yes, and both increases tyrosine phosphorylation of ZA proteins and opens the endothelial paracellular pathway through SFK activation.