Enamel is the hardest tissue in vertebrates. Ameloblasts are derived from epithelial cells and are responsible for enamel formation. They secrete enamel matrix components in which amelogenins are the major proteins, the biochemical properties of which are well known. However, little is known about the characteristics of ameloblasts themselves or about the functions of amelogenins. In this study, we developed a novel primary and secondary culture system for ameloblasts using a monoclonal antibody which recognized amelogenin (En3). The cell layer on dentine removed from rat mandibular incisors was isolated and cultured in low calcium, serum-free medium. Primary culture was performed on collagen-coated culture plates and typically, two types of cells appeared. One major type changed morphology after the addition of a high concentration of calcium to the medium. Expression of amelogenin was shown as cytoplasmic particles in these cells using En3. In the secondary culture, expression of amelogenins was also observed. In this system, the cells grew and maintained the expression of amelogenin for about 3 weeks.