In order to understand the mechanisms involved in tooth development it is important to define the timing for tissue-specific gene expression. A consequence of ameloblast cell differentiation is the sequential expression of tissue-specific genes whose products form the enamel extracellular matrix. The ameloblast phenotype has been characterized as consisting of two major classes of proteins: amelogenins and non-amelogenin proteins such as anionic enamel proteins (enamelins, tuft proteins, tuftelin, sulfated proteins) and enamel proteases. The postulated functions for the anionic enamel proteins are as nucleators for hydroxyapatite crystal formation while amelogenins control the crystal size, growth and orientation. While the amelogenins have been well characterized, detailed knowledge for anionic enamel proteins has been sparse. In the present study, we designed experiments to characterize one of the anionic enamel proteins from mouse molars, tuftelin, and to determine the timing of expression of this protein during molar tooth development. Our results showed the initial detection of tuftelin transcripts within proliferating inner enamel epithelial cells at very early stages of tooth development (13 days of embryonic development equivalent to the bud stage of tooth development). These data provide direct evidence that invalidates previous dogmas that enamel proteins were synthesized by polarized, non-dividing, fully differentiated ameloblast cells. In addition, tuftelin was found to be synthesized also by dental papilla mesenchyme cells suggesting that this protein is not enamel-specific. These data taken together open the possibility that the tuftelin present in the dentino-enamel junction could be secreted by both, preodontoblast cells and preameloblast cells. It might also suggest a possible different role for tuftelin than nucleator of hydroxyapatite crystals.