G protein-coupled receptors (GPCRs) of endothelial cells transmit diverse intracellular signals that regulate adherens junction (AJ) permeability. Increased endothelial permeability contributes to pathological processes such as inflammation, atherogenesis, and acute lung injury. Thrombin, a serine protease, and sphingosine-1-phosphate (S1P), a bioactive lipid, regulate endothelial barrier function by activating their respective GPCRs-the protease-activated receptor PAR(1) and the S1P receptor S1P(1)-which initiate intracellular signals that regulate AJ integrity and cytoskeleton organization. The distinct patterns of PAR(1) and S1P(1) signal transduction underlie the functional antagonism between thrombin and S1P. Evidence points to a role for activation of the S1P(1) receptor that is induced by PAR(1)-mediated signaling in the mechanism of AJ reannealing and endothelial barrier repair. Understanding the molecular basis of AJ integrity in the context of inflammation is important in developing novel anti-inflammatory therapeutics. This Review provides a working model for molecular mechanisms for the dual regulation of endothelial barrier function.