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 (S1P1-5receptors). S1P1and S1P2receptors, 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 S1P2signaling 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 S1P1and S1P2signaling regulates the homeostasis of microvascular permeability in the peripheral circulation and, thus, may affect total peripheral vascular resistance.