Effective therapies to limit the profound vascular leakage in inflammatory injury are severely limited. Our early translational investigations first suggested sphingosine 1-phosphate (S1P), a multifunctional lipid mediator and angiogenic factor, as an effective therapy for reducing vascular leakage in inflammatory lung injury. We have shown that S1P rapidly stimulates a Rac GTPase-driven signaling cascade leading to reorganization of the vascular endothelial cell cytoskeleton and enhanced junctional integrity, biophysical events which ultimately strengthen the EC barrier and decrease vascular permeability. Importantly, S1P administration to mice with endotoxin (LPS)-induced lung injury or to dogs in a lung injury model induced by intrabronchial LPS instillation, markedly improves oxygenation and reduces alveolar edema formation. As S1P is found in nano-micromolar quantities in plasma, the expression and dynamic responses of G-protein-coupled S1P receptors (S1PRs) serve to coordinate cellular responses to S1P. S1PR1, S1PR2, and S1PR3 are highly expressed in lung vascular endothelium, however, only ligation of S1PR1 reduces murine lung vascular permeability in response to LPS, ischemia/reperfusion, ventilator-induced injury and ionizing radiation. Furthermore, S1PR1 is critical to hepatocyte growth factor-, hyaluronan- and activated protein C-mediated EC barrier enhancement via S1PR1 transactivation. In contrast, ligation of S1PR3 induces Rho GTPase signaling to the cytoskeleton and increased lung permeability. We recently investigated the role of specific S1P/S1PR responses in inflammatory lung injury including dose- and delivery route-dependent (intratracheal vs intravenous) effects of S1P and selective S1PR receptor agonists and antagonists on vascular leakage in wild type and genetically-engineered mice in relevant murine models of inflammatory lung injury. We determined that direct intratracheal (IT) or intravenous (IV) administration of S1P, or a selective S1PR1 agonist (SEW- 2871) produces highly concentration-dependent barrier-regulatory responses in the murine lung. IT or IV administration of S1P or SEW-2871 at <0.3 mg/kg was protective against LPS-induced murine lung inflammation and permeability, however, IT delivery of S1P at 0.5 mg/kg (2hrs) resulted in significant alveolar-capillary barrier disruption (42% increase in BAL inflammatory protein) and produced rapid lethality when delivered at 2 mg/kg. Despite greater S1PR1 selectivity, IT-delivered SEW-2871 at 0.5 mg/kg also resulted in significant alveolar-capillary barrier disruption but was not lethal at 2 mg/kg. Consistent with S1PR1 regulation of alveolar/vascular barrier function, wild type mice pretreated with a S1PR1 antagonist, SB-649146, or S1PR1+/- mice exhibited loss of S1P/SEW-2871-mediated barrier protection following LPS challenge. Finally, our gene resequencing studies identified novel SNPs in genes involved in sphingolipid metabolism, S1P-mediated vascular signaling, and S1P-mediated barrier regulation which are potentially involved in susceptibility to ALI. These studies, which underscore the potential therapeutic effect of highly selective S1PR1 receptor agonists in reducing inflammatory lung injury, highlight the critical role of S1P delivery route, S1PR1 agonist concentration, and S1PR1 expression in target tissues.