Sphingosine is a biologically active derivative of sphingomyelin. It affects diverse cellular functions and its mechanism(s) of action is poorly defined. Tumor necrosis factor alpha (TNF alpha) has recently been shown to rapidly induce sphingomyelin turnover, implicating this metabolic pathway in TNF alpha signal transduction. Because TNF alpha is known to induce prostaglandin E2 (PGE2) production in human fibroblasts, we tested the effect of sphingosine on TNF alpha-induced PGE2 production. We found that sphingosine enhanced TNF alpha-induced PGE2 production by as much as 18-fold over TNF alpha alone. Sphingosine appeared to stimulate TNF alpha-induced PGE2 production independent of TNF alpha-mediated interleukin 1 (IL-1) production, because anti-IL-1 antibodies and IL-1 receptor antagonist protein (IRAP) did not inhibit TNF alpha-induced PGE2 production or the stimulatory effect of sphingosine. TNF alpha stimulated PGE2 production to the same degree in normal and protein kinase C (PKC) downregulated cells in the presence and absence of sphingosine, indicating that neither TNF alpha nor sphingosine require active PKC to elicit their respective effects. The sphingosine analogues stearylamine and stearoyl-D-sphingosine had little or no effect on TNF alpha-mediated PGE2 production, supporting a specific role for sphingosine in the activation process. Short-term (1 min) exposure of cells to sphingosine dramatically increased TNF alpha-induced PGE2 production. A potential mechanism by which sphingosine could increase TNF alpha-induced PGE2 production involves enhancement of phospholipase A2 (PLA2) and/or cyclooxygenase (Cox) activity, the rate-limiting enzymes in PGE2 production. We found that both TNF alpha and sphingosine alone enhanced these enzymatic activities, and that sphingosine additively increased the effect of TNF alpha on phospholipase A2 activity. It appears that sphingosine affects TNF alpha-induced PGE2 production via a mechanism that is independent of PKC involvement, and that sphingosine may function as an endogenous second messenger capable of modulating the responsiveness of the cell to external stimuli.