Recent studies point to a significant role of vasodilator-stimulated phosphoprotein (VASP) in the maintenance of endothelial barrier functions in vivo and in vitro. Moreover, it has been reported that VASP is required for activation of the small GTPase Rac 1. However, little is known whether VASP is involved in the regulation of cell adhesion molecules that are critical for maintenance of the endothelial barrier. Here we demonstrate that impaired barrier properties in VASP-deficient (VASP-/-) microvascular myocardial endothelial cells (MyEnd) correlated with both impaired integrin-mediated adhesion as revealed by laser tweezer trapping and reduced integrin-dependent cell migration. This was paralleled by reduction of focal adhesions at the cell periphery as well as of beta(1)-integrin and VE-cadherin cytoskeletal anchorage. Incubation of MyEnd VASP wt with RGD peptide to block interaction of integrins with extracellular matrix (ECM) reduced barrier properties and Rac 1 activity in wt endothelial monolayers mimicking the situation in VASP (-/-) cells under resting conditions. Moreover, cAMP-mediated Rac 1 activation was reduced under conditions of impaired integrin-mediated adhesion in wt cells and cAMP-induced increase in VE-cadherin cytoskeletal anchorage was abolished in VASP (-/-) endothelium. In summary, these data indicate that VASP is required for integrin-mediated adhesion which stabilizes endothelial barrier properties at least in part by facilitating Rac 1 activation.