We have previously found that an antigenic protein specific for C3H strain mouse (C3H strain-specific antigen, CSA) is identical to peptide-binding protein 74 (PBP74). PBP74/CSA is a novel member of the stress-70 protein family in mitochondria. In this study, mouse genomic clones encoding PBP74/CSA, including the 5'- and 3'-flanking regions of the gene, have been isolated and sequenced. The PBP74/CSA gene contained 17 exons interrupted by 16 introns. Two dimeric repeats of the consensus sequence of the heat-shock element are present in the 5'-flanking region of the PBP74/CSA gene. Moreover, the first intron is interrupted within the amino-terminal leader sequence, the pattern of which is similar to that of cytochrome c1 located in the mitochondria.