Scabrosin esters (SEs), which have been recently isolated from the lichen Xanthoparmelia scabrosa, belong to the epipolythiodioxopiperazine (ETP) class of secondary metabolites characterized by possession of a reactive disulfide bond. Colony forming assays show that these toxins are active against human tumor cell lines at nanomolar concentrations. Other members of the ETP class of toxins such as gliotoxin have been shown to induce apoptosis in cells, although the cellular target(s) of the ETP toxins is currently unknown. ETP toxins have been shown to inhibit a variety of enzymes via interaction with sensitive cysteine residues. Here we show that the typical scabrosin ester acetate butyrate induces early mitochondrial membrane hyperpolarization assessed by JC-1 staining accompanied by apoptotic cell death. The toxin lowers ATP in intact cells and inhibits the rate of ATP synthesis in permeabilzed cells. Comparison with the effects of the known ATP synthase inhibitor oligomycin B is consistent with ATP synthase as an early target in scabrosin ester-induced cell death.