Formation of calcified tissues is a well regulated process. In dentin, the odontoblasts synthesize several biomolecules that function as nucleators or inhibitors of mineralization. To identify genes that are odontoblast-specific, a subtractive hybridization technique was employed that resulted in the identification of a previously undescribed novel gene synthesized by the odontoblasts. Based on the nomenclature in our laboratory, this gene has been named dentin matrix protein 4 (DMP4). The protein encoded by mouse DMP4 cDNA contained 579 amino acids, including a 26-amino acid signal peptide. Analysis of the protein sequence demonstrated the presence of a Greek key calcium-binding domain and one conserved domain of unknown function in all the species examined thus far. Calcium binding property was confirmed by (45)Ca binding assays and the corresponding change in conformation by far-ultraviolet circular dichroism. Northern analysis demonstrated high expression levels of a single 3-kb mRNA transcript in tooth, whereas low expression levels were detected in other tissues. In situ hybridization analysis showed high expression levels of DMP4 in odontoblasts and low levels in osteoblasts and ameloblasts during tooth development. Gain and loss of function experiments demonstrated that DMP4 had the potential to differentiate mesenchymal precursor cells into functional odontoblast-like cells.