Sphingosine 1-phosphate (SPP) is a potent lipid mediator released upon cellular activation. In this report, pharmacological properties of the three G-protein-coupled receptors (GPCRs) for SPP, EDG-1, -3, and -5 are characterized using a Xenopus oocyte expression system, which lacks endogenous SPP receptors. Microinjection of the EDG-3 and EDG-5 but not EDG-1 mRNA conferred SPP-responsive intracellular calcium transients; however, the EDG-5 response was quantitatively much less. Co-expression of EDG-1 receptor with the chimeric Galphaqi protein conferred SPP responsiveness. Galphaqi or Galphaq co-injection also potentiated the EDG-5 and EDG-3 mediated responses to SPP. These data suggest that SPP receptors couple differentially to the Gq and Gi pathway. All three GPCRs were also activated by sphingosylphosphorylcholine, albeit at higher concentrations. None of the other related sphingolipids tested stimulated or blocked SPP-induced calcium responses. However, suramin, a polycyclic anionic compound, selectively antagonized SPP-activated calcium transients in EDG-3 expressing oocytes with an IC50 of 22 microM, suggesting that it is an antagonist selective for the EDG-3 GPCR isotype. We conclude that the three SPP receptors signal differentially by coupling to different G-proteins. Furthermore, because only EDG-3 was antagonized by suramin, variations in receptor structure may determine differences in antagonist selectivity. This property may be exploited to synthesize receptor subtype-specific antagonists.