Integrin-mediated endothelial cell-extracellular matrix adhesion plays a critical role in maintaining the structural integrity of microvascular walls. The aim of this study was to evaluate the impact of specific integrin extracellular domain binding to matrix fibronectin and vitronectin on the barrier function of intact microvascular endothelium. The apparent permeability coefficient of albumin was measured in isolated and perfused porcine coronary venules using a fluorescence ratioing technique with the aid of fluorescence microscopy. Inhibition of integrin binding to either fibronectin with GRGDdSP peptide or vitronectin with GPenGRGDSPCA peptide dose-dependently increased venular permeability by 2- to 3-fold. The effects were sustained for more than 60 min and were reversible upon clearance of the peptides. In contrast, the inactive control peptide GRADSP did not significantly affect venular permeability. Pretreatment of the venules with purified human fibronectin and vitronectin, respectively, prevented the hyperpermeability response to GRGDdSP and GPenGRGDSPCA. GRGDSP, a peptide that inhibits integrin binding to both fibronectin and vitronectin, produced an even higher permeability (4.5-fold) in venules than GRGDdSP or GPenGRGDSPCA alone, and the effect was blunted in vessels preincubated with both fibronectin and vitronectin. The results indicate the importance of integrin-matrix interaction in the physiological regulation of microvascular permeability. It is likely that both fibronectin and vitronectin binding to integrins contribute to the maintenance of endothelial barrier function in venules.