Therapeutic options for ameliorating the profound vascular permeability, alveolar flooding and organ dysfunction which accompanies acute inflammatory lung injury (ALI) remains limited. Extending our prior reports that intravenous (IV) administration of the sphingolipid angiogenic factor, sphingosine 1-phosphate (S1P), attenuates inflammatory lung injury and vascular permeability via ligation of S1PR1, we now determine 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 reduced S1P/SEW-2871-mediated barrier protection following LPS challenge. In contrast, S1PR2-/- KO mice as well as mice with reduced S1PR3 expression (via siS1PR3-containing nanocarriers) were protected against LPS-induced barrier disruption compared to controls. 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.