The ADP/ATP translocator is an abundant protein of the mitochondrial inner membrane, which in fungi and mammals is synthesized without a presequence. Here we report that the translocator from potato has an amino-terminal extension which may function in mitochondrial targeting. Several cDNA clones encoding the nucleotide sequence of the ADP/ATP translocator have been isolated from potato leaf and tuber cDNA libraries constructed in lambda phages. Only one class of cDNA clones was found but possibly different translocator genes are expressed in other tissues. High levels of transcripts for the translocator are found in all tissues analysed. Sequence determination of the complete insert of one of the clones reveals a long open reading frame of 1158 bp encoding a protein of 386 amino acids corresponding to a calculated molecular weight of 42 kDa. In contrast, the ADP/ATP translocator proteins from fungi and mammals are significantly smaller. Comparison of the Neurospora translocator with the potato protein shows about 75% sequence homology, being confined to the region after amino acid 85 of the potato polypeptide. Antibodies directed against the fungal translocator recognize a protein of 30 kDa in the inner membrane of potato mitochondria, suggesting that the mature protein has a similar size as the translocators from fungi and mammals. Thus, the additional segment of the potato ADP/ATP translocator forms an amino-terminal extension which may be involved in the import of the protein into plant mitochondria.