This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-α–mediated hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH) became sensitive to TNF-α, undergoing a time-dependent apoptotic cell death preceded by mitochondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A treatment rescued mGSH-depleted hepatocytes from TNF-α–induced cell death. In contrast, mGSH-depleted hepatocytes deficient in ASMase were resistant to TNF-α–mediated cell death but sensitive to exogenous ASMase. Furthermore, although in vivo administration of TNF-α or LPS to galactosamine-pretreated ASMase+/+ mice caused liver damage, ASMase–/– mice exhibited minimal hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylceramide synthetase inhibition on TNF-α–mediated apoptosis. This approach, which blunted glycosphingolipid generation by TNF-α, protected mGSH-depleted ASMase+/+ hepatocytes from TNF-α despite enhancement of TNF-α–stimulated ceramide formation. To further test the involvement of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase+/+ but not in ASMase–/– hepatocytes. However, treatment of ASMase–/– hepatocytes with exogenous ASMase induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-α–induced hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids.This work was supported in part by the Research Center for Liver and Pancreatic Diseases (P50 AA11999) and grant 1R21 AA014135-01 funded by the U.S. National Institute on Alcohol Abuse and Alcoholism; Plan Nacional de I+D grants SAF 99-0138, 2FD97-0988, and PM99-0166; and Fondo Investigaciones Sanitarias grant FISS 00-907. C. García-Ruiz is a Sistema Nacional de Salud investigator from the Fondo Investigaciones Sanitarias.Peer reviewed