Sphingosine 1-phosphate (S1P) increases intracellular Ca²⁺concentration in many cell types, but the signaling mechanism remains uncertain. The recent identification of three closely related seven-transmembrane domain receptors for S1P, termed Edg1, H218, and Edg3, support the extracellular ligand role of S1P and allowed examination of Ca²⁺ responses mediated specifically by each receptor subtype. To substantiate each subtype in S1P-induced Ca²⁺ responses and to study the transductional mechanisms, we applied the aequorin luminescence method and the fura-2 fluorescence method in two transfected mammalian cell systems. We showed that H218 and Edg3 were capable of mediating S1P-induced mobilization of intracellular Ca²⁺ when transiently transfected in human TAg-Jurkat T cells. Ca²⁺ responses mediated by Edg1 in TAg-Jurkat cells required coexpression of the G_qi5 chimeric G protein that links G_i-coupled receptors to G_q. When H218 and Edg3 were stably expressed in rat HTC4 hepatoma cells, S1P induced Ca²⁺ responses with nanomolar EC₅₀ values. Edg3, but not H218, elicited a sustained influx of extracellular Ca²⁺. The coincident formation of inositol phosphates and the complete inhibition of Ca²⁺responses by the phospholipase C inhibitor U73122 indicated that H218 and Edg3 mobilized Ca²⁺ through activation of phospholipase C. Partial inhibition of Ca²⁺ responses and inositol phosphates formation by pertussis toxin implied that H218 and Edg3 transduce phospholipase C activation and Ca²⁺ responses only partially through G_i proteins. Although these results did not dismiss that S1P may function as an intracellular second messenger in other settings, they definitively proved that S1P can mobilize Ca²⁺ as an extracellular ligand for G protein-coupled receptors.