Objectives: Protection of endothelial barrier function is inevitable for the recovery of the reperfused heart. Previously, we have shown that connexin 43 (Cx43)-dependent ATP release from endothelial cells during reperfusion protects the endothelial barrier against reperfusion injury. The aim of the present study was to investigate the signaling mechanism regulating Cx43-dependent ATP release from EC during reoxygenation. Methods: Cultured endothelial cells from porcine aorta were exposed to 40 min of hypoxia (Po2<10 mmHg) followed by reoxygenation (Po2=140 mmHg), while control cells were exposed to normoxia for the same period of time. ATP release (luciferin- luciferase assay), Cx43 content, and phosphorylation state at Ser 368 (Western blot) were determined. Results: Cx43 content was reduced by siRNA silencing. Reoxygenation- induced ATP-release was accompanied by a transient increase of Cx43 phosphorylation. In presence of PKA inhibitor H-89 (20mM), KT-5720 (100nM), as well as PKA inhibitor peptide ([6- 22amide]-NH2, 100nM) reoxygenation-induced ATP release was abolished. Accordingly, reoxygenation-induced Cx43 phosphorylation was prevented. However, activation of the cAMP/PKA signaling pathway by 8-Bromo cAMP (1mM), Sp-8- pCPT cAMPS (10 mM), and 6-Bnz-cAMP (10 mM) at the onset of reoxygenation enhanced the reoxygenation-induced ATP release. Silencing of Cx43 by siRNA abolished reoxygenation-induced ATP release and also abolished the effect of cAMP/PKA activation on ATP release in these cells. Conclusion: The data of the present study demonstrate for the first time that in endothelial cells reoxygenation-induced ATP release by Cx43 is cAMP/PKA-dependent. Strengthening of this endogenous mechanism by direct activation of cAMP/PKA pathway could represent a new therapeutic strategy to prevent acute endothelial barrier failure during reperfusion-injury.