Sphingosine-1-phosphate (Sph-1-P) is the initial product of catabolism of sphingosine by sphingosine kinase and is cleaved by Sph-1-P lyase to a fatty aldehyde and ethanolamine phosphate. This phosphorylated sphingoid base is not only an intermediary catabolite, but also a bioactive lipid with important functions, including stimulation of cell proliferation in Swiss 3T3 fibroblasts and inhibition of tumor cell motility. In the present study, we examined functional roles of Sph-1-P in human platelets. Sph-1-P induced platelet shape change and aggregation reactions, although it failed to elicit secretion. Sphingosine, ceramide, sphingomyelin, and N,N-dimethylsphingosine did not mimic the positive effects of Sph-1-P on platelets. Subthreshold concentrations of Sph-1-P and weak platelet agonists such as adenosine diphosphate (ADP) and epinephrine synergistically elicited aggregation, which may be important for efficient amplification of platelet activation. Sph-1-P induced intracellular Ca²⁺ mobilization and the dose-response for Ca²⁺ release correlated closely with the concentration required for induction of shape change. On addition of [³H]sphingosine to intact platelets, the label was rapidly converted to Sph-1-P, and subsequently to ceramide and sphingomyelin. Interestingly, the Sph-1-P formed was specifically released into medium on stimulation of platelets with physiologic agonists. The amount of Sph-1-P in platelets, as measured by its conversion into radiolabeled N-acetyl-Sph-1-P, was 1.4 nmol/ 10⁹ cells and was about four times higher than the mass of Sph present. When compared by mole percent Sph-1-P/ phospholipid, the value for platelets is over 10 times higher than that for neutrophils. Our results suggest that Sph-1-P, rapidly converted from sphingosine, abundantly stored in platelets, and released on the cell activation, may play a physiologic role in thrombosis, hemostasis, and the natural wound-healing processes.