An anomalous dentino–enamel junction (DEJ), manifested by delamination of the enamel layer, was reported in enamelysin [matrix metalloproteinase-20 (MMP-20)] knockout (KO) mice. To better understand the possible role of MMP-20 in the formation of the DEJ, we performed transmission electron microscopy (TEM) studies of the DEJ at early stages of tooth morphogenesis in KO mice. Our TEM analysis revealed that in the incisors from KO mice the mantle dentin is hypomineralized at the onset of enamel mineralization. At this early stage, TEM revealed no apparent differences in nascent aprismatic enamel between the KO mice and the controls. Hypomineralized mantle dentin was also observed in the incisors from KO mice, as assessed by back-scattered SEM at the secretory and early maturation stages, but not in the late-maturation stage, suggesting that the mineralization of mantle dentin is not completely arrested, but rather postponed. Histological studies indicate that the organic content in the initial enamel layer remains very high throughout amelogenesis. These results imply that MMP-20 is involved in the regulation of mineralization in mantle dentin and demonstrate the complex nature of DEJ formation. They also suggest that the structural and functional properties of the DEJ are determined during the initial mineralization stages.