SPRR3, a member of the SPRR family of cornified envelope precursor proteins, is expressed in oral and esophageal epithelia, where it is strictly linked to keratinocyte terminal differentiation. This gene is characterized by intragenic duplications that have created the characteristic proline-rich repeats in the coding sequence, an alternative noncoding exon, and a 200-bp polypyrimidine tract in the promoter region. Mutational analysis of the promoter region and transient transfection in normal human keratinocytes showed that in addition to the polypyrimidine tract, multiple regulatory elements are involved in differentiation-specific expression. These elements include a high-affinity Ets binding Site bound by ESE-1, an AP-1 site (TRE) recognized by the Jun/Fos family of transcription factors, and an ATF/CRE bound by Jun/Fos and ATF factors. The repositioning of the SPRR3 Ets binding site during evolution has a major effect on the relative contribution of this site to promoter activity.