Sphingosine-1-phosphate (S1P) regulates cell growth and survival, migration and adhesion in many cell types. S1P is generated by sphingosine kinases (SphKs), and dephosphorylated by phosphatases or cleaved by S1P lyase. Extracellular S1P activates specific G protein-coupled receptors while intracellular S1P can mobilize Ca2+ from thapsigargin-sensitive stores. Here, we have studied Ca2+ signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P lyase. In these cells, S1P and sphingosine concentrations were elevated about 6-fold and 2-fold, respectively, as measured by liquid chromatography/tandem mass spectrometry. Measurements with fura-2-loaded cells in suspension revealed that resting [Ca2+]i was elevated and agonist-induced [Ca2+]i increases were augmented in S1P lyase-deficient MEFs both in the presence and absence of extracellular Ca2+. Importantly, [Ca2+]i increases and Ca2+ mobilization induced by the SERCA inhibitor, thapsigargin, were augmented, indicating enhanced Ca2+ storage in S1P lyase-deficient MEFs. Measurements with single cells expressing the calmodulin-based Ca2+ sensor, cameleon, revealed that at least two cell types could be distinguished in both MEF cell populations, one with a rapid and transient [Ca2+]i increase and the other with a slower and prolonged [Ca2+]i elevation upon stimulation with thapsigargin. The area under the time course of thapsigargin-induced [Ca2+]i increases, reflecting overall Ca2+ release, was significantly increased by more than 50% in both rapidly and slowly responding S1P lyase-deficient cells. It is concluded that elevated concentrations of S1P and/or sphingosine lead to enhanced Ca2+ storage and elevated basal [Ca2+]i. S1P metabolism thus plays a role not only in acute Ca2+ mobilization but also in long-term regulation of Ca2+ homeostasis.