--- - |2+ Abstract Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and ATPγS can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to adenosine, ATPγS‐induced PKA activation is accompanied by a modest, but significant decrease in cAMP levels supporting the existence of an unconventional cAMP‐independent pathway of PKA activation. Furthermore, ATPγS‐induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP, but is instead dependent upon protein kinase A‐anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase‐targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS‐induced increases in transendothelial electrical resistance (TER). Adenosine‐induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP‐dependent manner. In summary, ATPγS‐induced enhancement of the EC barrier is EPAC1‐independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP‐independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation. This article is protected by copyright. All rights reserved - 'Journal of Cellular Physiology, Volume 0, Issue ja, -Not available-. '