Calicheamicin thetaII is a member of the enediyne class of antitumor antibiotics that bind to DNA and induce apoptosis. These compounds differ, however, from conventional anticancer drugs as they bind in a sequence-specific manner noncovalently to DNA and cause sequence-selective oxidation of deoxyriboses and bending of the DNA helix. Calicheamicin is clinically employed as immunoconjugate to antibodies directed against, for example, CD33 in the case of gemtuzumab ozogamicin. Here, we show by the use of the unconjugated drug that calicheamicin-induced apoptosis is independent from death-receptor/FADD-mediated signals. Moreover, calicheamicin triggers apoptosis in a p53-independent manner as shown by the use of p53 knockout cells. Cell death proceeds via activation of mitochondrial permeability transition, cytochrome c release and activation of caspase-9 and -3. The overexpression of Bcl-x(L) or Bcl-2 strongly inhibited calicheamicin-induced apoptosis. Knockout of Bax abrogated cell death after calicheamicin treatment. Thus, the activation of mitochondria and execution of cell death occur through a fully Bax-dependent mechanism. Interestingly, caspase inhibition by the pancaspase-inhibitor zVAD-fmk interfered with mitochondrial activation by calicheamicin. This places caspase activation upstream of the mitochondria and indicates that calicheamicin-triggered apoptosis is enhanced through death receptor-independent activation of the caspase cascade, that is, an amplification loop that is required for full activation of the mitochondrial pathway.