The growth of cells in vitro can provide useful models for investigating their behaviour and improving our understanding of their function in vivo. Although the developmental regulation of enamel matrix formation has been comprehensively analysed, the detailed cellular characteristics of ameloblasts remain unclear because of the lack of a system of long-term in vitro culture. Therefore, the establishment of odontogenic epithelial cell lines has taken on a new significance. Here, we report on a novel porcine odontogenic epithelial cell-culture system, which has permitted serial culture of these cells. Epithelial cells were harvested from third molar tooth buds in the fresh mandibles of 6-month-old pigs, and seeded on dishes in D-MEM containing 10% FBS. Before the cells reached confluence, the medium was changed to LHC-9 to select the epithelial cells. When trypsinized epithelial cells were plated together with 3T3-J2 cells as a feeder layer, the epithelial cells grew from single cells into colonies. The colonies then expanded and became confluent, and could be sub-cultured for up to 20 passages. The long-term culture cells expressed mRNA for amelogenin and ameloblastin, as well as enamelysin (MMP-20), which is a tissue-specific gene product unique to ameloblasts. These results show that the system is capable of sustaining the multiplication of odontogenic epithelial cells with the characteristics of ameloblasts.