CYM-5541 is a full agonist, able to reach the maximum level of ERK phosphorylation that is observed with S1P. CYM-5541 has an EC ₅₀ of between 72 and 132 nM and exhibits exquisite selectivity over other S1P receptor subtypes: S1P1 EC ₅₀ >10 μM, S1P2 EC ₅₀ >50 μM, S1P4 EC ₅₀ >50 μM, and S1P5 EC ₅₀ >25 μM. CYM-5541 also shows selectivity over a large panel of protein targets, with no significant activities, in the Ricerca profiling panel of 55 GPCRs, ion channels, and transporters. CYM-5541 allowed us to identify an allosteric site where F263 is a key gate-keeper residue for its affinity and efficacy. The novel allosteric hydrophobic pocket may account for the S1P3 selectivity of CYM-5541.