Adenosine triphosphate (ATP) is a physiologically relevant agonist released by activated platelets with complex effects mediated via activation of P2 receptors. Although ATP-induced endothelial cell (EC) production of prostacyclin and nitric oxide is recognized, its effects on EC barrier function and vascular permeability remain poorly characterized. Although we recently reported EC barrier enhancement and cytoskeletal activation by ATP the mechanisms involved remains unclear. We have previously identified activation of the small guanosine triphosphatase (GTPase) Rac as key to EC barrier augmentation (simvastatin, sphingosine 1-phosphate) and therefore examined the role of Rac in ATP-induced EC barrier enhancement. Human pulmonary artery EC, grown to confluence overlying gold microelectrodes, were treated with ATP (1-100 μM) or vehicle and transendothelial electrical resistance (TER) measured (n = 3). EC were also grown to ˜ 50% confluence and treated with ATP (10 μM, 1-20 min) and the p21 (Rac) binding domain of PAK1 fused to GST used to precipitate activated Rac GTP complexes followed by Western blotting for Rac and quantification by densitometry. Finally, EC were transfected with Rac siRNA and TER again measured in response to ATP (10 μM). ATP induced rapid, dose-dependent barrier enhancement (TER) with a maximal effect appreciable at 30 min (˜ 38% increase, 10 μM), persisting at 2 h. ATP-treated EC (10 μM) demonstrated a significant increase in Rac activation relative to control with a maximal effect (˜ 4-fold increase) at 10 min (n = 3, p<0.05). Finally, ATP-induced barrier enhancement was markedly reduced by silencing of Rac (˜ 50%) relative to controls. Together our results suggest that ATP-mediated barrier protection is dependent on Rac activation and contribute to our understanding of EC activation and barrier regulation by platelets at sites of injury.