Sphingosine-1-phosphate (SPP), a metabolite of sphingolipids, has been implicated as a second messenger in cell growth regulation and signal transduction via calcium mobilization from internal stores. This study shows that SPP mobilizes intracellular calcium in U937 cells and demonstrates for the first time the ability of SPP to activate the transcription factor NF-kappa B in these cells. Furthermore, calcium release from the internal stores by thapsigargin (TG), an inhibitor of the endoplasmic reticulum Ca2+ pump, was associated with activation of NF-kappa B. Moreover, we have shown that while an intracellular calcium chelator BAPTA/AM was able to inhibit both SPP- and TG-induced NF-kappa B activation, it had no effect on TNF-induced NF-kappa B activation. In addition, SPP-induced NF-kappa B activation was blocked both by cyclosporin A, known to inhibit calcineurin phosphatase activity, and by the antioxidant butylated hydroxyanisole. These observations suggest that intracellular calcium mobilization is required for SPP-induced NF-kappa B activation, which may involve calcineurin- and redox-dependent mechanisms.