The two major dentin matrix proteins, dentin sialoprotein and dentin phosphoprotein have been shown to be expressed as a single large transcript termed dentin sialophosphoprotein (DSPP). These non-collagenous matrix proteins, identified biochemically by their unique physical-chemical properties, are specific cleavage products of a large parent acidic phosphorylated protein (pI 4.0). Previous studies have shown expression of dentin sialoprotein at the protein level by ameloblasts. The purpose of this study was to determine the temporal-spatial pattern of DSPP expression during amelogenesis. In situ hybridization and immunohistochemistry were performed on sections of developing mouse molars. These data were correlated with RT-PCR analysis of in vitro enamel organ epithelium monolayer cell cultures enriched for ameloblasts. Our data indicates initial expression of the DSPP transcripts and protein during early ameloblast differentiation prior to the secretory phase when the majority of the enamel matrix is formed. Ameloblasts appear to tightly down-regulate DSPP transcription as enamel matrix formation is up-regulated. These data demonstrate DSPP expression during amelogenesis is under highly controlled developmental regulation. Therefore, DSPP may have a primary role in the initial mineralization events of both enamel and dentin, acting as a potential nucleator of hydroxyapatite crystal formation.