We have cloned and characterized the genomic structure of the mouse gene for prolyl oligopeptidase that is mapped to chromosome 10B2-B3. The gene is about 92 kilobases in size and contains 15 exons. All exon-intron junction sequences conform to the GT/AG rule. Comparison with the presumed domain structures of the mouse prolyl oligopeptidase indicates that the propeller domain of the enzyme is encoded by exons 3-10, whereas the catalytic domain is encoded by exons 1-3 and 10-15. The catalytic triad residues are encoded by two exons (Ser(554) on exon 13 and His(680) and Asp(642) on exon 15). The 5'-flanking region of the mouse prolyl oligopeptidase gene has structural features found in housekeeping gene promoters, including a GC-rich segment and an absence of TATA and CAAT boxes. A primer extension assay showed the presence of multiple sites for the initiation of transcription. Transient transfection analysis demonstrated that the 5'-flanking region of the gene can direct efficient expression in COS1 cells. Deletion studies revealed that the downstream 125-base pair sequence of the region is required for promoter activity in the cells.