The protein (PTN) encoded by the pleiotrophin (PTN) gene belongs to a recently described family of heparin-binding cytokines whose expression is temporally and spatially regulated during development. We have now isolated genomic clones of the human PTN gene, characterized its promoter region, determined its transcription initiation site(s), and established functional activity of the PTN promoter. A fragment -550/+191 that contains a CAAT box, no apparent TATA box, and four consensus sites for the binding of MyoD is sufficient to provide optimal promoter activity. A serum response element is found at -559 to -568. We also have identified the human PTN gene on chromosome 7, band q33 and the mouse Ptn gene on chromosome 6, respectively. The data thus identify and characterize the 5' end of the PTN gene and its promoter region, suggest potential regions that may contribute to the regulation of its transcriptional activity, and localize the PTN gene in human and mouse chromosomes.