Mast cell stimulation via IgE receptors causes activation of multiple processes, including Ca(2+) mobilization, granule exocytosis, and outward trafficking of recycling endosomes to the plasma membrane. We used fluorescein-conjugated cholera toxin B (FITC-CTxB) to label GM(1) in recycling endsomes and to monitor antigen-stimulated trafficking to the plasma membrane in both fluorimeter and imaging-based assays. We find that the sphingosine derivatives D-sphingosine and N,N'-dimethylsphingosine effectively inhibit this outward trafficking response, whereas a quarternary ammonium derivative, N,N',N″-trimethylsphingosine, does not inhibit. This pattern of inhibition is also found for Ca(2+) mobilization and secretory lysosomal exocytosis, indicating a general effect on Ca(2+)-dependent signaling processes. This inhibition correlates with the capacity of sphingosine derivatives to flip to the inner leaflet of the plasma membrane that is manifested as changes in plasma membrane-associated FITC-CTxB fluorescence and cytoplasmic pH. Using a fluorescently labeled MARCKS effector domain to monitor plasma membrane-associated polyphosphoinositides, we find that these sphingosine derivatives displace the electrostatic binding of this MARCKS effector domain to the plasma membrane in parallel with their capacity to inhibit Ca(2+)-dependent signaling. Our results support roles for plasma membrane polyphosphoinositides in Ca(2+) signaling and stimulated exocytosis, and they illuminate a mechanism by which D-sphingosine regulates signaling responses in mammalian cells.