Ceramide has been implicated as a second messenger in intracellular signaling pathways leading to apoptosis in nonhepatic cells. To determine whether ceramide can mediate hepatocyte apoptosis, the cytotoxicity of ceramide was determined in rat hepatocytes. The rat hepatocyte cell line, RALA255-10G, and primary rat hepatocytes were completely resistant to toxicity from 10 to 100 μmol/L C2 ceramide. Resistance was not the result of a failure to take up ceramide, because ceramide treatment did cause nuclear factor-κB (NF-κB) activation. Because ceramide may mediate cell death from tumor necrosis factor α (TNF-α), the ability of RNA synthesis inhibition and NF-κB inactivation to sensitize hepatocytes to ceramide toxicity was examined. RALA hepatocytes were sensitized to ceramide toxicity by coadministration of actinomycin D (ActD). Cell death occurred by apoptosis as determined by the presence of morphological evidence of apoptosis, caspase activation, poly(ADP-ribose) polymerase (PARP) degradation, and DNA hypoploidy. Despite the induction of apoptosis associated with caspase activation, cell death from ActD/ceramide was not blocked by caspase inhibition. Inhibition of NF-κB activation also sensitized RALA hepatocytes to ceramide toxicity, but to a lesser extent than for TNF-α. Thus, unlike many nonhepatic cell types, rat hepatocytes are resistant to cell death from ceramide because of the transcriptionally dependent up-regulation of a protective gene(s). The ability of ActD and NF-κB inactivation to sensitize RALA hepatocytes to ceramide toxicity suggests that ceramide may act as a downstream mediator of TNF-α toxicity.