Nucleobindin, a Ca2+-binding protein, has been previously identified within the nucleus and endoplasmic reticulum, and in association with the Golgi membrane. In addition, nucleobindin has been shown to be a minor constituent of bone extracellular matrix and has been postulated to play a role in mineralization. In the current investigation, we report the expression and localization of nucleobindin within odontoblasts and the dentin matrix. Nucleobindin mRNA transcripts were detected in the tooth, and in situ hybridization analysis substantiated the findings, showing nucleobindin expression within mature odontoblasts and within the cells of surrounding developing alveolar bone. Western blot analysis of tooth protein extracts demonstrated the presence of a 63 kDa protein, which showed immunologic affinity for a rat nucleobindin peptide antibody. The distribution of the protein was shown in mature odontoblasts by using immunohistochemistry. Moreover, immunogold labeling of nucleobindin and subsequent ultrastructural analysis demonstrated a similar pattern of distribution. Nucleobindin was identified within odontoblast cellular compartments: the nucleus, endoplasmic reticulum, and mitochondria. Of interest, nucleobindin localization was observed within the surrounding dentin extracellular matrix, and immunogold labeling was shown to accumulate with tissue development toward the cusp. The study clearly demonstrated the presence of nucleobindin within dental tissues. In consideration of the known functional properties of nucleobindin, it may be postulated that nucleobindin may contribute to the accumulation and transport of Ca2+ ions to the mineralization front prior to hydroxyapatite deposition.