Ceramide is generated by the hydrolysis of membrane sphingomyelin by sphingomyelinase and is implicated in multiple signaling pathways, including those regulating differentiation, inflammation and immune responses. Excess formation of prostaglandin E 2 (PGE 2 ) is thought to increase susceptibility to infection, rheumatoid arthritis and inflammation, including periodontal diseases. We investigated the inhibitory effect of C 2 -ceramide, a short-chain ceramide analog, on the PGE 2 -stimulated accumulation of cAMP in human gingival fibroblasts. In human gingival fibroblasts pre-treated with C 2 -ceramide for 18 h, the PGE 2 -stimulated accumulation of cAMP was reduced, but an inactive C 2 -ceramide analog had no such effect. The accumulation of cAMP induced by EP2 and EP4 receptor agonists (ONO-AE1-259 and ONO-AE1-329, respectively) was inhibited in cells treated with C 2 -ceramide. However, treatment with C 2 -ceramide had no effect on the expression of mRNAs encoding the EP2 and EP4 receptors. Accumulation of cAMP could be induced by cAMP-elevating agents (forskolin, isobutylmethylxanthine and mastparan) but was not reduced by treatment with C 2 -ceramide. These observations suggest that C 2 -ceramide attenuates PGE 2 receptor function and consequently inhibits the accumulation of cAMP in human gingival fibroblasts.Prostaglandins are lipid mediators that are involved in many physiological and pathophysiological processes. The excess formation of prostaglandin E 2 (PGE 2 ) has been described in a number of conditions characterized by increased susceptibility to infection, rheumatoid arthritis and inflammation, including periodontal diseases (8,17,25,29). PGE 2 mediates its biological functions via binding to four types of membrane-bound, G protein-coupled receptors, termed E prostanoid (EP)1 to EP4 (6, 22). EP receptors stimulated by ligand binding activate different signal transduction pathways. Activation of the EP1 receptor raises intracellular Ca 2+ levels. Activation of EP2 and EP4 receptors increases intracellular cAMP levels by activating adenylate cyclase via G s proteins. Activation of the EP3 receptor reduces or increases cAMP levels by activating inhibitory G (G i ) or stimulatory G (G s ) proteins depending on the particular splice variant expressed by the cell (15). In human gingival fibroblasts, PGE 2 inhibited the DNA synthesis and proliferation (1, 37), and downregulated intercellular adhesion molecule-1 expression by cAMP-dependent signaling pathways (24). Therefore, cAMP is thought to be the main intracellular second messenger for response to PGE 2 , and is the crucial modulator of the functional activity of inflammation. Ceramide, an intracellular second messenger in the sphingomyelin transmembrane signaling pathway, can be generated by the cleavage of sphingomyelin by sphingomyelinase or by de novo biosynthesis by ceramide synthase (14). Increases in intracellular ceramide have been reported in many