Human hepatocytes usually are resistant to TNF-α cytotoxicity. In mouse or rat hepatocytes, repression of NF-κB activation is sufficient to induce TNF-α-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IκBα (Ad5IκB), which almost completely blocks NF-κB activation,> 80% of the cells survived 24 h after TNF-α stimulation. Here, we report that TNF-α activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N, N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-α in Ad5IκB-infected Huh-7 and Hc cells. TNF-α-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-α-induced apoptosis. Although Akt has been reported to activate NF-κB, DMS and LY 294002 failed to prevent TNF-α-induced NF-κB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-κB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-κB but also SphK and PI3K/Akt are involved in the signaling pathway (s) for protection of human hepatocytes from the apoptotic action of TNF-α and probably FasL.