Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are major causes of acute respiratory failure associated with high morbidity and mortality. Although ALI/ARDS pathogenesis is only partly understood, pulmonary endothelium plays a major role by regulating lung fluid balance and pulmonary edema formation. Consequently endothelium-targeted therapies may have beneficial effects in ALI/ARDS. Recently, attention has been given to the therapeutic potential of purinergic agonists and antagonists for the treatment of cardiovascular and pulmonary diseases. Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface P2Y receptors. We previously described ATP-induced endothelial cell (EC) barrier enhancement via a complex cell signaling, and hypothesized endothelial purinoreceptors activation to exert anti-inflammatory barrier-protective effects. To test this hypothesis we used a murine model of ALI induced by intratracheal administration of endotoxin/lipopolysaccharide (LPS), and cultured pulmonary EC. The non-hydrolyzed ATP analogue ATPS (50-100 µM final blood concentration) attenuated inflammatory response with decreased accumulation of cells (48%, P<0.01), and proteins (57%, P<0.01) in bronchoalveolar lavage, reduced neutrophil infiltration and extravasation of Evans blue albumin dye into lung tissue. In cell culture model, ATPS inhibited junctional permeability induced by LPS. These findings suggest that purinergic receptor stimulation exerts a protective role against ALI by preserving integrity of endothelial cell-cell junctions.