Bcl-2 is an integral, intracellular membrane protein that prevents cells from undergoing apoptosis in response to a variety of cell death signals. It negatively regulates the activation of Caspase-3, which functions as effector of mammalian cell death pathways. Overexpression of Bcl-2 inhibits the caspase activities and apoptosis. A microbial secondary metabolite, Tetrocarcin A (TC-A), was identified as an inhibitor of the anti-apoptotic function of Bcl-2. Apoptosis could be induced in cell lines that overexpressed Bcl-2 or Bcl-X_L when the cells were treated with anti-Fas antibody,tumor necrosis factor α, staurosporine, or Bax, in addition to TC-A. TC-A showed selectivity against the pro-apoptotic Bcl-2 family members, in that cells overexpressing CrmA or dominant-negative FADD could not undergo apoptosis with TC-A treatment. In Bcl-2-overexpressing cell lines, TC-A inhibited mitochondrial functions regulated by Bcl-2, resulting in Fas-triggered mitochondrial transmembrane potential loss and cytochrome c release. Inhibition of the mitochondrial functions of Bcl-2 and, thereby, its anti-apoptotic effect could serve as useful pharmacological targets. Thus, TC-A should serve as an archetype for specific inhibitors of Bcl-2 functions.