Strain SPB-1 is a temperature-sensitive Chinese hamster ovary cell mutant defective in sphingoid base biosynthesis. To examine the effects of sphingolipid deficiency on a glycosyl phosphatidylinositol-anchored protein, we constructed transfectants expressing mouse CD14 from the SPB-1 and the wild type cells. When the cells were precultured in a sphingolipid-deficient medium at 39 degrees C, CD14 expressed in the SPB-1 cells was hypersensitive to phosphatidylinositol-specific phospholipase C (PI-PLC), compared with that in the wild type cells. Moreover, after exposure of the cells to fumonisin B1, an inhibitor of ceramide synthase, CD14, even in the wild type cells, became hypersensitive to PI-PLC. The PI-PLC hypersensitivity in the SPB-1 cells was almost completely suppressed when the cells were cultured in the presence of exogenous sphingosine or sphingomyelin. On the other hand, the hypersensitivity induced by fumonisin B1 was suppressed by exogenous sphingomyelin but not by sphingosine. The suppression by exogenous glucosylceramide was partial under both conditions. Likewise, CD14 molecules in membranes prepared from the sphingolipid-deficient cells were more sensitive to PI-PLC than those from the control cells. These results indicated that a deficiency in cellular complex sphingolipids caused the PI-PLC hypersensitivity of CD14, suggesting the interaction of CD14 with sphingolipids in membranes.