Apoptosis mediated by anticancer drugs may involve activation of death-inducing ligand/receptor systems such as CD95 (APO-1/Fas), cleavage of caspases, and perturbance of mitochondrial functions. We investigated the sequence of these events in SHEP neuroblastoma cells transfected with Bcl-2 or Bcl-X_l using two different drugs, namely, doxorubicin (Doxo), which activates the CD95/CD95 ligand (CD95-L) system, and betulinic acid (Bet A), which does not enhance the expression of CD95 or CD95-L and which, as shown here, directly targets mitochondria. Apoptosis induced by both drugs was inhibited by Bcl-2 or Bcl-X_l overexpression or by bongkrekic acid, an agent that stabilizes mitochondrial membrane barrier function, suggesting a critical role for mitochondria. After Doxo treatment, enhanced CD95/CD95-L expression and caspase-8 activation were not blocked by Bcl-2 or Bcl-X_l and were found in cells with a mitochondrial transmembrane potential (ΔΨ_m) that was still normal (ΔΨ_m^high cells). In marked contrast, after Bet A treatment, caspase-8 activation occurred in a Bcl-2- or Bcl-X_l-inhibitable fashion and was confined to cells that had lost their ΔΨ_m (ΔΨ_m^low cells). Mitochondria from cells treated with either Doxo or Bet A induced cleavage of both caspase-8 and caspase-3 in cytosolic extracts. Thus, caspase-8 activation may occur upstream or downstream of mitochondria, depending on the apoptosis-initiating stimulus. In contrast to caspase-8, cleavage of caspase-3 or poly(ADP-ribose)polymerase was always restricted to ΔΨ_m^low cells, downstream of the Bcl-2- or Bcl-X_l-controlled checkpoint of apoptosis. Cytochrome c, released from mitochondria undergoing permeability transition, activated caspase-3 but not caspase-8 in a cell-free system. However, both caspases were activated by apoptosis-inducing factor, indicating that the mechanism of caspase-8 activation differed from that of caspase-3 activation. Taken together, our findings demonstrate that perturbance of mitochondrial function constitutes a central coordinating event in drug-induced cell death.