Mitochondrial ADP/ATP translocase is an abundant component of the inner membrane. It carries ATP from the matrix into the intermembrane space and transports ADP back. Clones coding for two different but related forms of the protein have been characterized from bovine cDNA libraries. The corresponding genes are referred to as T1 and T2 and they are expressed at different levels in bovine tissues. The bovine cDNAs have been used to isolate clones from a human genomic library that contain the human T1 and T2 genes. Two nucleotide sequences of 9756 and 8625 base-pairs have been determined and they contain the transcribed regions of the human T1 and T2 genes which cover 4.2 and 5.9 kb of the human genome, respectively (1 kb = 10(3) base-pairs). Both genes are split into four exons. The introns in each gene are at exactly equivalent locations and interrupt sequences coding for segments of the protein that are thought to be extramembranous loops linking transmembrane segments. The proteins encoded in the genes differ in 32 amino acids out of 297, and in common with other ADP/ATP translocases, neither has a processed mitochondrial import sequence. The human T1 and T2 genes are members of a larger gene family that includes a third expressed gene T3 and also at least two spliced pseudogenes. Other studies have shown that T3 is expressed in liver and HeLa cells, and different levels of transcripts of T1 have been found in various tissues. A notable feature of the T1 and T2 genes, that may influence their expression, is that "CpG-rich islands" are associated with their 5' ends. That of the T2 gene contains numerous potential sites for binding the mammalian transcription factor SP1, but no TATA or CCAAT sequences are evident near to its 5' end, although these latter features are associated with the human T1 gene. The two DNA sequence also contain many short interspersed repetitive sequences including 11 Alu repeats, and a novel element about 236 base-pairs in length, which is repeated in a six-fold tandem array in intron B of the T2 gene.