To elucidate the physiologic role of sphingolipid-derived products as signaling molecules, we analyzed the levels of endogenous sphingosine (Sph) derivatives in human platelets. When the platelets were stimulated with thrombin or 12-O-tetradecanoylphorbol 13-acetate, neither ceramide formation nor sphingomyelin hydrolysis was observed, which suggests that the sphingomyelin cycle may not be an essential part of the signaling pathway under these conditions. In contrast, Sph was found to increase in platelets upon stimulation. The level of Sph 1-phosphate, which is formed from Sph by the action of Sph kinase, was not affected under our conditions. Although it has been established that Sph inhibits protein kinase C, which regulates the functional responses of the platelets, Sph levels which exert an inhibitory effect on protein kinase C cannot be attained under physiological conditions (without exogenous Sph). Considering the stimulation of the synthesis of Sph by the physiological agonist thrombin, we speculate that Sph is a signaling molecule of physiological importance in platelets, but protein kinase C may not be its target.