Hypoxia/reoxygenation (H/R) causes cellular injury and death. The cell death pathways induced by H/R remain incompletely understood. H/R can induce Bid and Bax mitochondrial translocation and cytochrome c release. Using mouse lung endothelial cells (MLEC), we examined the role of Bcl-X(L), an anti-apoptotic Bcl-2-related protein, in H/R-induced cell death. The expression of Bcl-X(L) protected MLEC against H/R-induced cell death by blocking Bax and Bid translocation and inhibiting mitochondrial cytochrome c release. Bcl-X(L) expression inhibited caspase-8 cleavage and death-inducing signal complex (DISC) formation in plasma membrane. By isolating mitochondrial, nuclear, and Golgi fractions, we found that H/R induced DISC formation in these organelles. Bcl-X(L) expression inhibited DISC formation in the nuclear and Golgi fractions relative to LacZ-infected controls. In contrast, DISC formation was elevated in the mitochondrial fraction of Bcl-X(L)-infected cells. GRASP65, a Golgi-associated protein, physically associated with Fas and caspase-8; Bcl-X(L) expression decreased these associations. Bcl-X(L) expression also up-regulated FLIP, a caspase-8 inhibitor. In conclusion, Bcl-X(L) may inactivate caspase-8 by decreasing DISC formation in the plasma membrane, nucleus, and Golgi complex while diverting DISC formation to the mitochondria. The inhibitory effects of Bcl-X(L) on DISC formation may play significant roles in protecting endothelial cells from H/R-induced cell death.