Ribophorin I is a type I transmembrane glycoprotein characteristic of the rough portions of the endoplasmic reticulum where it is thought to play a role in the cotranslational insertion of nascent polypeptides. A rat ribophorin I cDNA was used to isolate four overlapping genomic clones from a rat EMBL3 genomic library. Restriction mapping, Southern blotting, and DNA sequencing showed that these clones, spanning approximately 21 kilobases of chromosomal DNA, include the entire ribophorin I gene, as well as 15 kilobases (kb) of upstream sequences. Southern blotting analysis of DNA from a panel of mouse-Chinese hamster cell hybrids demonstrated that the ribophorin I gene is located on mouse chromosome six. The ribophorin I gene contains 10 exons, seven of which encode the luminal domain of the polypeptide. Exon 8 encodes the trans-membrane domain and small portions of the flanking luminal and cytoplasmic domains. Exons 9 and 10 encode the remainder of the cytoplasmic domain, and the latter includes the 3'-untranslated portion of the mRNA. Six closely spaced transcription start sites located 3 to 24 base pairs upstream from the initiation codon were identified by primer extension analysis and S1 mapping. The sequence of a 1.3-kb region upstream of the cap sites was determined and found to contain three GC-rich potential Sp1-binding sites beginning at -14, -24, and -91 base pairs (bp), two octamer-like sequences at -233 and -1248 bp, and a CAAT-like box at -41 bp. The possible roles of these elements in regulating expression of the ribophorin gene in all cells and in differentiated cell types characterized by a well developed rough endoplasmic reticulum is discussed.