Besides its role as a putative second messenger releasing Ca2+ from intracellular stores, sphingosine-1-phosphate (SPP) has recently been identified as an extracellularly acting ligand activating a high affinity G protein-coupled membrane receptor in various cell types. Since SPP can be released from activated platelets, we examined in the present study whether endothelial cells express receptors for SPP and related sphingolipids. In bovine aortic endothelial cells loaded with fura-2, addition of SPP caused a rapid and transient increase in intracellular Ca2+ concentration ([Ca2+]i), amounting to maximally about 230 nM. Removal of extracellular Ca2+ revealed that SPP-induced [Ca2+]i elevations were due to both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Pretreatment of the cells with pertussis toxin inhibited the SPP-induced increase in [Ca2+]i by 83%, in line with the previously reported involvement of G proteins of the Gi/o family in SPP signalling in other cell types. In contrast to other [Ca2+]i-elevating agonists, e.g., ATP and bradykinin, SPP did not activate phospholipase C in bovine aortic endothelial cells, suggesting the involvement of a novel, unidentified signalling pathway in SPP-induced release of intracellular Ca2+. Furthermore, SPP also did not cause activation of either phospholipase D or A2. Out of various related sphingolipids studied, only sphingosylphosphorylcholine (SPPC) induced a similar maximal increase in [Ca2+]i as SPP, and its effect was also fully pertussis toxin-sensitive. However, the potencies of the two sphingolipids to increase [Ca2+]i differed by more than two orders of magnitude, with the EC50 values being 0.8 nM and 260 nM for SPP and SPPC, respectively. These results identify SPP and SPPC as novel and potent endothelial agonists, inducing calcium signalling by activation of a Gi/o protein-coupled receptor(s). Given the recently reported release of SPP from thrombin-activated platelets, SPP may represent a novel mediator of platelet-endothelial cell interactions.