Inositol 1,4,5-trisphosphate (IP 3) has long been recognized as a second messenger for intracellular Ca 2+ mobilization. Recently, sphingosine 1-phosphate (S1P) has been shown to be involved in Ca 2+ release from the endoplasmic reticulum (ER). Here, we investigated the role of S1P and IP 3 in antigen (Ag)-induced intracellular Ca 2+ mobilization in RBL-2H3 mast cells. Antigen-induced intracellular Ca 2+ mobilization was only partially inhibited by the sphingosine kinase inhibitor dl- threo-dihydrosphingosine (DHS) or the IP 3 receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB), whereas preincubation with both inhibitors led to complete inhibition. In contrast, stimulation of A 3 adenosine receptors with N 5-ethylcarboxamidoadenosine (NECA) caused intracellular Ca 2+ mobilization that was completely abolished by 2-APB but not by DHS, suggesting that NECA required only the IP 3 pathway, while antigen used both the IP 3 and S1P pathways. Interestingly, however, inhibition of IP 3 production with the phospholipase C inhibitor U73122 completely abolished Ca 2+ release from the ER induced by either stimulant. This suggested that S1P alone, without concomitant production of IP 3, would not cause intracellular Ca 2+ mobilization. This was further demonstrated in some clones of RBL-2H3 cells excessively overexpressing a β isoform of Class II phosphatidylinositol 3-kinase (PI3KC2β). In such clones including clone 5A4C, PI3KC2β was overexpressed throughout the cell, although endogenous PI3KC2β was normally expressed only in the ER. Overexpression of PI3KC2β in the cytosol and the PM led to depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2), resulting in a marked reduction in IP 3 production. This could explain the abolishment of intracellular Ca 2+ mobilization in clone 5A4C. Supporting this hypothesis, the Ca 2+ mobilization was reconstituted by the addition of exogenous PI(4,5)P 2 in these cells. Our results suggest that both IP 3 and S1P contribute to FcɛRI-induced Ca 2+ release from the ER and production of IP 3 is necessary for S1P to cause Ca 2+ mobilization from the ER.