We have been investigating the molecular mechanisms underlying pathophysiological regulation of microvascular permeability on isolated venules and cultured venular endothelial monolayers. Physiological approaches have been employed in combination with molecular analyses to probe the signal transduction pathways leading to enhanced microvascullar permeability. A newly developed technique of protein transfection into cells and intact microvessels enables the correlation of fullctional reactions and signaling events at the molecular level in a direct and specific fashion. The results indicate that inflammatory mediators increase microvascular permeability via intracellular signaling pathways involving the activation of phospholipase C, cytosolic calcium, protein kinase C, nitric oxide synthase, guanylate cyclase, and protein kinase G. In response to the signaling stimulation, complex biochemical and conformational reactions occur at the endothelial structural proteins. Specifically, myosin light-chain activation-mediated myosin light-chain phosphorylation can result in cell contraction. VE-cadherin and beta-catenin phosphorylation may induce dissociation of the junctional proteins and their connection to the cytoskeleton, leading to a loose or opened intercellular junction. Focal adhesion phosphorylation and redistribution further provide an anchorage support for the conformational changes in the cells and at the cell junction. The three processes may act in concert to facilitate the flux of fluid and macromolecules across the microvascular endothelium.