Aims: Activation of the cAMP signalling leads to attenuation of the thrombin-induced hyperpermeability. One of the mechanisms underlying this protective effect is the inactivation of endothelial contractile machinery, a major determinant of endothelial barrier function, mainly via activation of myosin light chain phosphatase (MLCP). To date, the mechanisms of cAMP-mediated MLCP activation are only partially understood. Here the contribution of two cAMP effectors, PKA and Epac, in the regulation of endothelial contractile machinery and barrier function was studied. Methods: Endothelial contractile machinery and barrier function were analyzed in cultured HUVEC. 8-CPT-cAMP, 6-Bnz-cAMP and Forskolin (FSK) were used to activate Epac, PKA or adenylyl cyclase, respectively. The cells were challenged by inflammatory mediator thrombin to inhibit MLCP via RhoA/Rock pathway. Results: Activation of either PKA or Epac could partially block the thrombin-induced barrier failure. Simultaneous activation of PKA and Epac had additive effects, which were comparable to FSK. Activation of PKA but not Epac inhibited thrombin-induced phosphorylation of MLC and the MLCP regulatory subunit MYPT1, in part due to inhibition of the RhoA/Rock pathway. FSK caused activation of the MLCP catalytic subunit PP1 via dephosphorylation and dissocoation of the PP1 inhibitory protein CPI-17. FSK blunted the thrombin-induced CPI-17 phosphorylation, CPI- 17/PP1 complex formation and PP1 inactivation. Down-regulation of CPI-17 by siRNA attenuated thrombin-induced hyperpermeability and abolished the antagonistic effect of the PKA activator, whereas the Epac activator retained its antagonistic effect. Conclusion: cAMP/PKA regulates the endothelial barrier via inhibition of contractile machinery mainly by activation of MLCP via inhibition of CPI-17 and RhoA/Rock. The permeability lowering effect of the cAMP/Epac pathway is independent of CPI-17.