The dicyclohexylcarbodi-imide-reactive proteolipid is a membrane subunit of mitochondrial ATP synthase. In cows it is encoded by two different nuclear genes known as P1 and P2. These genes are expressed in a tissue-specific fashion which reflects the embryonic origin of the tissues. The proteins that they encode are synthesized in the cytosol, and are precursors of the proteolipid that have different mitochondrial import sequences of 61 and 68 amino acids respectively. By use of gene-specific probes derived from the bovine P2 cDNA, regions containing corresponding parts of the bovine P2 gene have been isolated from a bovine genomic library, and their DNA sequences and those of flanking and intervening regions have been determined. The sequence contains four exons, which represent the cDNA sequence, spread over 3.8 kb of the bovine genome. Two of the introns are in the DNA sequence coding for the mitochondrial import sequence, and a third intron is in a sequence encoding an extramembranous structure between the two putative transmembrane alpha-helical domains of the mature proteolipid. An Alu-type repetitive element was detected at the extreme 5' end of the sequence. The bovine P1 and P2 genes for the dicyclohexylcarbodimide-reactive proteolipid of ATP synthase are members of a multiple gene family that also contains many pseudogenes. The bovine P1 gene has not been isolated, but two distinct P1 pseudogenes have been cloned and their DNA sequences have been determined. Both of them contain 'in-phase' stop codons and frame-shift mutations, and one of them bears the hallmarks of retroposition; it has no introns, it contains a poly(A) tract at its 3' end and it is flanked by direct DNA sequence repeats. The second P1 pseudogene is very unusual. It appears to be derived from a partially processed transcript and contains an intervening DNA sequence of 861 bp that corresponds in position with an intron in the human P1 gene. This pseudogene also could have been introduced by retroposition since its sequence is flanked by short direct repeats. However, it does not contain a poly(A) tract at its 3' end. An alternative, but less likely, explanation is that rather than being a retroposon, this sequence arose by duplication of an expressed gene at a time when it had only one intron.