We investigated the effects of retinoic acids on mitochondrial permeability transition (MPT) measured as changes in rhodamine 123 fluorescence from both isolated heart mitochondria and HeLa cells. We report that all-trans-retinoic acid (atRA), 9-cis-retinoic acid, and 13-cis-retinoic acid induce a drop in mitochondrial membrane potential in isolated mitochondria. The atRA effect was done through the induction of MPT because it was dependent on Ca(2+), in a synergic mechanism, and inhibited by cyclosporin A (CsA). Furthermore, atRA also opened MPT in vivo, because treatment of HeLa cells with atRA results in a CsA-sensitive drop of mitochondrial membrane potential. We demonstrated for the first time that retinoic acids inhibit adenine nucleotide translocase (ANT) activity in heart and liver mitochondria. Kinetic studies revealed atRA as an uncompetitive inhibitor of ANT. Photoaffinity labeling of mitochondrial proteins with [3H]atRA demonstrated the binding of a 31-kDa protein to atRA. This protein was identified as ANT because the presence of carboxyatractyloside, a specific ANT inhibitor, prevented labeling. The specific photolabeling of ANT was also prevented in a concentration-dependent manner by nonlabeled atRA, whereas palmitic acid was ineffective. This study indicates that specific interaction between atRA and ANT takes place regulating MPT opening and adenylate transport. These observations establish a novel mechanism for atRA action, which could control both energetic and apoptotic mitochondrial processes in situations such as retinoic acid treatment.