Sphingosine 1-phosphate (S1P) is a bioactive lipid that binds to and activates a family of GPCRs, S1P1-5 (also known as EDG receptors). Interactions between S1P and its receptors mediate cytoskeletal rearrangement and cell migration, with functional consequences in angiogenesis, lymphocyte trafficking, and smooth muscle development (Anliker and Chun, 2004). S1P1 (Edg-1) signals exclusively through Gi, whereas S1P2 (Edg-5) and S1P3 (Edg-3) activate Gi, Gq and G12/13 (Windh et al., 1999). Although S1P1 and S1P3 promote cell migration, S1P2 inhibits cell migration in several cell types; these opposing functions appear to result from differences in the ability of each receptor to activate Gi (Sugimoto et al., 2003). Studies with knockout mice indicate that S1P2 and S1P3 have redundant functions in maintaining vascular integrity during embryonic development (Kono et al., 2004). In addition, S1P3 regulates immune responses by contributing to endothelial barrier function in splenic marginal zones (Girkontaite et al., 2004). The cloned human S1P3 -expressing cell line is made in the Chem-1 host, which supports high levels of recombinant S1P3 expression on the cell surface and contains high levels of the promiscuous G protein Gα15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between S1P3 and its ligands.