An acute increase in the sphingomyelin content of a plasma membrane-enriched fraction of 3T3-L1 cells was produced by incubation of the cells with 0.1 microM dexamethasone for 4 hr. Dexamethasone also stimulated the activity of the pathway of sphingomyelin synthesis by utilizing the phosphorylcholine of phosphatidylcholine as a donor to ceramide to synthesize the phospholipid (phosphatidylcholine:ceramide cholinephosphotransferase). Dexamethasone-stimulated increase in the utilization of 14C-labeled choline of phosphatidylcholine for the synthesis of sphingomyelin was inhibited by the addition of cycloheximide to the incubation. Therefore, it appears that corticosteroid stimulation of new protein synthesis was required to produce the effect. An increase in the enzymatic pathway by 83% and of the sphingomyelin content of the plasma membrane-enriched fraction by 50% after incubation with dexamethasone for 4 hr demonstrates the rapidity with which the hormone can produce considerable remodeling of the membrane. The increase in the synthetic pathway in the plasma membrane-enriched fraction was sufficient to account for the measured increase in sphingomyelin. It appears likely that the large increase in membrane sphingomyelin could contribute significantly to the many demonstrated effects of corticosteroids upon membrane processes, including transport, receptors, and enzymatic activity.