Different isoforms of the adenine nucleotide translocase (ANT) are expressed in a tissue-specific manner. It was assumed that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be differently distributed within the membrane structures that constitute the peripheral inner membrane or the crista membrane. To discriminate between ANT originating from peripheral or from cristal inner membranes we made use of the fact that complexes between porin, the outer-membrane pore protein, and the ANT can be generated. Such complexes between porin and the ANT in the peripheral inner membrane were induced in rat heart mitochondria and isolated from rat brain and kidney. Using ANT-isotype-specific antibodies and sequence analysis of the N-terminal end, it was discovered that the peripheral inner membrane contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively ANT-2. Cyclophilin was co-purified with the porin-ANT complexes, whereas it was absent in the crista-derived ANT. This suggested that ANT-1 might have a higher affinity for cyclophilin. This specific intra-mitochondrial distribution of the two ANT isotypes and cyclophilin D suggests specific functions of the peripheral and crista-forming parts of the inner membrane and the two ANT isotypes therein.