Sphingosine-1-phosphate (S1P) regulates various molecular and cellular events in cultured endothelial cells, such as cytoskeletal restructuring, cell-extracellular matrix interactions, and intercellular junction interactions. We utilized the venular leakage model of the cremaster muscle vascular bed in Sprague-Dawley rats to investigate the role of S1P signaling in regulation of microvascular permeability. S1P signaling is mediated by the S1P family of G protein-coupled receptors (S1P_1-5 receptors). S1P₁ and S1P₂ receptors, which transduce stimulatory and inhibitory signaling, respectively, are expressed in the endothelium of the cremaster muscle vasculature. S1P administration alone via the carotid artery was unable to protect against histamine-induced venular leakage of the cremaster muscle vascular bed in Sprague-Dawley rats. However, activation of S1P₁-mediated signaling by SEW2871 and FTY720, two agonists of S1P₁, significantly inhibited histamine-induced microvascular leakage. Treatment with VPC 23019 to antagonize S1P₁-regulated signaling greatly potentiated histamine-induced venular leakage. After inhibition of S1P₂ signaling by JTE-013, a specific antagonist of S1P₂, S1P was able to protect microvascular permeability in vivo. Moreover, endothelial tight junctions and barrier function were regulated by S1P₁- and S1P₂-mediated signaling in a concerted manner in cultured endothelial cells. These data suggest that the balance between S1P₁ and S1P₂ signaling regulates the homeostasis of microvascular permeability in the peripheral circulation and, thus, may affect total peripheral vascular resistance.