Lipid rafts, composed of sphingolipids, are critical to Toll-like receptor 4 (TLR4) assembly during lipopolysaccharide (LPS) exposure as a result of phosphokinase C (PKC)-zeta activation. However, the mechanism responsible for these events remains unknown.We determined whether LPS-induced TLR4 assembly and activation are dependent on the sphingolipid metabolite ceramide, produced by acid sphingomyelinase following the initial binding of LPS to CD14.Cultured THP-1 cells were stimulated with LPS, exogenous C(2) ceramide, or both. Selected cells were pretreated with the acid sphingomyelinase inhibitor imipramine or CD14 neutralizing antibody.Exposure to LPS led to activation of acid sphingomyelinase, production of ceramide, phosphorylation of PKCzeta, and assembly of the TLR4 receptor within lipid rafts. This was followed by activation of the MAPK family of products and the liberation of tumor necrosis factor-alpha. Pretreatment with imipramine or CD14 blockade was associated with attenuation of all of these LPS-induced events. Simultaneous treatment with C(2) ceramide and LPS reversed all the inhibitory effects induced by imipramine, but not those associated with CD14 blockade.Assembly and activation of the TLR4 receptor following LPS binding to CD14 requires the production of ceramide by acid sphingomyelinase.