Atractyloside (Atr) binds to the adenine nucleotide translocator (ANT) and inhibits ANT-mediated ATP/ADP exchange on the inner mitochondrial membrane. In addition, Atr can trigger opening of a non-specific ion channel, within the ANT-containing permeability transition pore complex (PTPC), which is subject to redox regulation and inhibited by cyclosporin A (CsA). Here we show that the cytotoxic effects of Atr, both in vivo and in vitro, are determined by its capacity to induce PTPC opening and consequent mitochondrial membrane permeabilization (MMP). Thus, the Atr-induced MMP and death of cultured liver cells are both inhibited by CsA as well as by glutathione (GSH) and enhanced by GSH depletion. Similarly, the hepatorenal toxicity of Atr, assessed in vivo, was reduced by treating mice with CsA or a diet rich in sulfur amino acids, a regime which enhances mitochondrial GSH levels. Atr injection induced MMP in hepatocytes and proximal renal tubular cells, and MMP was reduced by either CsA or GSH. Acetaminophen (paracetamol)-induced acute poisoning was also attenuated by CsA and GSH, both in vitro and in vivo. Altogether these data indicate that PTPC-mediated MMP may determine the hepatorenal toxicity of xenobiotics in vivo.