Fas (CD95)-mediated apoptosis is an essential mechanism for the maintenance of homeostasis, and disruption of this death pathway contributes to many human diseases. The cell survival protein kinase Akt/protein kinase B (PKB) is a known regulator of apoptosis, but its role in Fas-mediated cell death and its regulatory mechanisms are unclear. In this study, we show that stimulation of the Fas receptor by its ligand (FasL) induces rapid phosphorylation of Akt/PKB and a parallel increase in cell apoptosis in epidermal Cl41 cells. Inhibition of PI3K/Akt by dominant-negative overexpression of PI3K (Deltap85) and Akt (Akt-T308A/S473A) protects the cells from apoptosis, indicating an unexpected proapoptotic role of PI3K/Akt in the Fas signaling process. Treatment of the cells with pharmacological inhibitors of PI3K, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-1 (LY294002), similarly inhibits FasL-induced apoptosis and Akt/PKB phosphorylation, indicating that PI3K is an upstream mediator of Akt/PKB and is involved in Fas-mediated cell death. Electron spin resonance studies show that FasL treatment induces rapid generation of reactive oxygen species, and inhibition of ROS by antioxidants effectively inhibits Akt/PKB signaling, suggesting that FasL activation of Akt/PKB is redox sensitive. In cells transfected with dominant-negative PI3K/Akt, Fas expression is down-regulated, but FLIP expression is unaffected. Reporter gene and mRNA expression assays show that FasL activates fas transcriptional activity and this effect is inhibited by PI3K/Akt suppression. Together, our results indicate that the PI3K/Akt, in addition to its normal prosurvival role, also plays an apoptotic role in Fas-mediated cell death through a mechanism that involves transcriptional activation of Fas receptor.