Sphingosine 1-phosphate (S1P) is a bioactive phospholipid released by activated blood platelets that serves to influence heart rate, coronary artery caliber, endothelial integrity, lung epithelial integrity and lymphocyte recirculation through five related high affinity G-protein coupled receptors. S1P3 receptor couples promiscuously to Gi, Gq, and G12/13 proteins, and its tissue distribution is widespread. Bradycardia and hypertension are clearly associated with S1P3 activation and its expression patterns in cardiac tissue. S1P3 on dendritic cells has been identified as a major exacerbating factor for mortality during sepsis by playing a role in the critical linkage of inflammation and coagulation pathways downstream of the thrombin cascade. Understanding the contributions of the individual S1P receptors has been limited by the unavailability of selective modulators for the 5 receptor subtypes. In the pilot phase of the Molecular Libraries Probe Production Centers Network (MLPCN), The Scripps Research Institute Molecular Screening Center (SRIMSC) reported four low micromolar agonist probes for S1P3: ML003, ML004, ML005 and ML006. The current report describes the development of ML249, a submicromolar allosteric agonist probe for S1P3. ML249 resulted from high-throughput screening using a cell-based assay employing a Chinese Hamster Ovary (CHO) cell line stably transfected with human S1P3 receptor, nuclear factor of activated T-cell-beta lactamase (NFAT-BLA) reporter construct, and the Gα16 pathway coupling protein, followed by medicinal chemistry efforts. ML249 activates S1P3 receptor with an EC50 of 72.3 nM–132 nM, and is inactive as an agonist against other members of the receptor family S1P1 (EC50 >10 μM), S1P2 (EC50 >50 μM), S1P4 (EC50 >50 μM), and S1P5 (EC50 >25 μM). Evidence is presented showing that ML249 is an allosteric agonist; it does not compete with physiological ligand S1P for binding to S1P3. In addition, ML249 is nontoxic to U2OS cells, with a CC50 >10 μM. ML249 was submitted to Ricerca Biosciences, LLC for target profiling against a panel of receptors, transporters, and ion channels; the data suggest that compound ML249 is generally inactive against a broad array of off-targets and does not likely exert unwanted effects. ML249 is the first submicromolar, completely selective S1P3 receptor agonist to be identified, and as an allosteric agonist promises to facilitate determination of key receptor interactions that would not otherwise be possible.