DMP1 is an acidic phosphorylated protein with the spatial and temporal expression that is largely restricted to bone and tooth tissues. The biological function of DMP1 is associated with biomineralization of bone, cartilage and tooth development. To study the cell-specific expression of DMP1, a 2,512 bp upstream segment of the human gene was isolated and characterized. A series of progressive deletions of the human DMP1 5′ flanking sequence were ligated to the luciferase reporter gene, and their promoter activities examined in transfected human osteoblast-like (MG-63) and dental pulp (HDP-D) cells that express DMP1 and hepatic (HepG2) and uterine (HeLa) cells lacking DMP1 expression. A critical cis-regulatory element located between nt -150 and -63 was found to act as a specific silencer responsible for the negative regulation of DMP1 in HepG2 and HeLa cells. The transcriptional activity of this element in MG-63 and HDP-D cells had a 5-7-fold increase than that observed in HepG2 and HeLa cells. Electrophoretic mobility shift assays (EMSAs) showed that a 6-bp DNA sequence in this element was bound by two nuclear factors that are expressed at high levels in HepG2 and HeLa versus MG-63 and HDP-D cells. Competitive assays by EMSAs suggest that the 6-bp core DNA sequence, AG(T/C)C(A/G)C, is a novel DNA-protein binding site and conserved with high identity in reported DMP1 promoters for all species. Furthermore, point mutations of the core sequence caused a marked increase of DMP1 promoter activity in HepG2 and HeLa cells. We speculate that this silencing cis-element may play a critical role in the regulation of DMP1 cell-specific expression. © 2004 Wiley-Liss, Inc.