Several reports support the concept that bile acids may be cytotoxic during cholestatic disease process by causing mitochondrial dysfunction. Here we report additional data and findings aimed at a better understanding of the involvement of the permeability transition pore (PTP) opening in bile acids toxicity. The mitochondrial PTP is implicated as a mediator of cell injury and death in many situations. In the presence of calcium and phosphate, chenodeoxycholic acid (CDCA) induced a permeability transition in freshly isolated rat liver mitochondria, characterized by membrane depolarization, release of matrix calcium, and osmotic swelling. All these events were blocked by cyclosporine A (CyA) and the calcium uniporter inhibitor ruthenium red (RR). The results suggest that CDCA increases the sensitivity of isolated mitochondria in vitro to the calcium-dependent induction of the PTP.