Highly purified adenylate translocase (ANT) from rat heart mitochondria was functionally reconstituted as ATP/ADP exchange carrier in asolectin/cardiolipin vesicles. The ANT preparations used were free of porin, cyclophilin D, and Bax as analysed immunologically and by activity measurements. After pre-loading the ANT-containing proteoliposomes with ATP, malate or AMP, a gradual release of the trapped compounds by increasing the external Ca2+ concentrations could be demonstrated. N-Methyl-Val-4-cyclosporin did not inhibit the Ca2+ dependent release of internal substances from ANT liposomes. This inhibitor was found to be specific for the mitochondrial permeability transition pore (MTP) in intact mitochondria or reconstituted MTP-like protein complexes (e.g. hexokinase, porin, ANT complex). However, ADP in concentrations > 20 microM inhibited the liberation of internal compounds, while in contrast, atractyloside (30 microM) and HgCl2 (5 microM) both induced permeability of the ANT-containing liposomes resulting in a release of trapped substances. These results strongly suggest that ANT itself is capable to adopt a pore-like structure under conditions known to induce the permeability transition in mitochondria.