Sphingosine-1-phosphate (S1P) receptor agonists are novel immunosuppressive agents. The selectivity of S1P1 against S1P3 is strongly correlated with lymphocyte sequestration and minimum acute toxicity and bradycardia. This study describes molecular modeling, site-directed mutagenesis, and affinity studies exploring the molecular basis for selectivity between S1P1 and S1P3 receptors. Computational models of human S1P1 and S1P3 receptors bound with two nonselective agonists or two S1P1-selective agonists were developed based on the X-ray crystal structure of bovine rhodopsin. The models predict that S1P1 Leu276 and S1P3 Phe263 contribute to the S1P1/S1P3 selectivity of the two S1P1-selective agonists. These residues were subjected to site-directed mutagenesis. The wild-type and mutant S1P receptors were expressed in Chinese hamster ovary cells and examined for their abilities to bind to and be activated by agonists in vitro. The results indicate that the mutations have minimal effects on the activities of the two nonselective agonists, although they have dramatic effects on the S1P1-selective agonists. These studies provide a fundamental understanding of how these two receptor-selective agonists bind to the S1P1 and S1P3 receptors, which should aid development of more selective S1P1 receptor agonists with immunosuppressive properties and improved safety profiles.