Sequential and reciprocal interactions between the oral ectoderm and neural crest-derived mesenchyme are responsible for tooth development. During dentin formation, there are three components that are necessary for proper mineralization, namely, collagen which forms a scaffold, noncollagenous proteins that can specifically bind to the collagen template and function as a mineral nucleator and crystalline calcium phosphate deposited in an ordered manner. It is well established that noncollagenous proteins play an important role during mineralized tissue formation. Here we demonstrate by in situ hybridization techniques that the noncollagenous dentin matrix proteins 1, 2 (DMP1, 2) and dentin sialoprotein (DSP) have characteristic temporal and spatial expression patterns within odontogenic tissues during dentin mineralization. DMP1, DMP2 and DSP mRNA are expressed in the odontoblasts at specific and overlapping time points and are thus presumably used for different functions during dentin formation. In developing rat incisors and molars, high levels of expression of DMP2 mRNA were seen in polarized odontoblasts and preameloblasts, while DSP mRNA was expressed at significantly lower levels and was expressed by highly differentiated odontoblasts. However, their expression was continuously maintained during the mineralization of the organic matrix. In the adult rats, DMP2 and DSP mRNA was also detected in the osteoblasts. The expression of DMP1 mRNA was found to coincide with the start of the mineral nucleation process and gradually decreased during the maturation of the mineralized matrix during odontogenesis. In this study, we have also correlated the expression of these proteins relative to the presence of type I collagen and calcium phosphate crystals. Thus, the temporal and spatial differences between DMP1, DMP2 and DSP might implicate a direct demonstration of the functional difference between these three genes during calcified tissue formation.