Accumulating evidence points to a significant role of vasodilator-stimulated phosphoprotein (VASP) in the maintenance of endothelial barrier functions. We have recently shown that impaired barrier functions in VASP-deficient microvascular myocardial endothelial cells (MyEnd VASP(-/-)) correlated with decreased Rac 1 activity. To further test the hypothesis that VASP is involved in regulation of Rac 1 activity, we studied cAMP-dependent Rac 1 activation. Both inhibition of Rac 1 activation by NSC-23766 and inhibition of PKA by PKI completely blunted the efficacy of forskolin/rolipram (F/R)-mediated cAMP increase to stabilize barrier functions as revealed by measurements of transendothelial resistance (TER). Because these results indicate that PKA/Rac 1 activation is important for barrier stabilization, we tested this signaling pathway in VASP(-/-) cells. We found that F/R and isoproterenol reduced permeability measured as FITC-dextran flux across VASP(-/-) monolayers, but not below baseline levels of wild-type cells (WT). Moreover, cAMP-mediated Rac 1 activation was reduced to approximately 50% of WT levels, and both PKA inhibition by PKI and PKA anchoring via A kinase anchoring peptides (AKAPs) by HT31 almost completely abolished Rac 1 activation in VASP(-/-) and WT endothelium. Accordingly, HT31 significantly reduced F/R-mediated TER increase in WT cells and completely blocked the protective effect of cAMP on endothelial barrier properties. Together, our data underline the significant role of cAMP-mediated Rac 1 activation for endothelial barrier stabilization and demonstrate that both AKAP-mediated PKA anchoring and VASP are required for this process.