We previously identified the gerbil gene (gPres) that encodes prestin, the putative motor protein responsible for outer hair cell (OHC) electromotility. Here we report the cloning and characterization of the complete genomic structure of the mouse Prestin (mPres) gene. We performed 5'- and 3'-RACE to determine the size and identity of the full-length mRNA transcript. The mPres gene encodes a protein 96% identical to the gPres gene product. The prestin open reading frames are 91% identical at the nucleotide level. Using an antibody raised against the N-terminus of gerbil Prestin, we observed mPrestin expression by immunofluorescence in the lateral membrane of mouse OHCs and found no detectable expression elsewhere in the organ of Corti. On the basis of the available genomic sequence from mouse Chromosome (Chr) 5, we concluded that the mPres gene is centromerically related to and resides within 19 kb of, the Reln gene. We were also able to characterize the exon/intron junctions of mPres by using cDNA/genomic sequence comparisons, as well as exon-exon PCR and sequencing. The mPres gene has 18 exons that encode protein and two exons in the 5' UTR. A CpG island, located at the start of exon 1, is a potential transcription start site. Sequence analysis of the ~500 bp upstream from exon 1 revealed multiple potential transcription factor binding sites, including both TATA and GC boxes, as well as other regulatory-element binding sites.