Calcium mobilization through antigen receptors, including high-affinity IgE receptors (Fc epsilon RI), is thought to be mediated by inositol-1,4,5-trisphosphate production (InsP(3))(1-4). Here we show that antigen clustering of Fc epsilon RI on the rat mast-cell line (RBC 2H3) activates a sphingosine kinase (SK) and produces sphingosine-l-phosphate (S1P), an alternative second messenger for intracellular calcium mobilization, The sphingosine analogue, D-L-threo-dihydrosphingosine (DHS), inhibits the SK enzyme competitively with a dissociation constant, K-i, of 5 to 18 mu M. This inhibition substantially suppresses the Fc epsilon RI-mediated calcium signal, but leaves intact the syk tyrosine kinase activation and the small InsP(3) production. The entire InsP(3)-dependent pathway activated by a transfected G-protein coupled receptor, used here as a positive control, also remained intact. Thus Fc epsilon RI principally utilizes a SK pathway to mobilize calcium.