Dental papilla mesenchymal cells differentiate into odontoblasts through epithelial-mesenchymal interactions. However, the mechanism by which enamel epithelial cells affect the differentiation of dental mesenchymal cells remains unknown. Alkaline phosphatase (ALPase) is a marker for odontoblast-like differentiation, because odontoblasts show much higher ALPase activity than dental undifferentiated mesenchymal cells. The continuously growing rabbit incisor is a good model for the epithelial-mesenchymal interaction during odontogenesis. In the present study, we isolated and maintained rabbit incisor-derived epithelial cells and rabbit incisor pulp-derived fibroblastic cells, and examined the effect of epithelial cells on ALPase activity in fibroblastic cells. Epithelial cells were stained with anti-cytokeratin 5 and 8 antibodies and showed the expression of tuftelin mRNA. In separate cultures of epithelial cells or fibroblastic cells, ALPase activity and mRNA levels were very low, but were upregulated in co-cultures of epithelial and fibroblastic cells. Histochemical analysis found high ALPase activity in fibroblastic cells close to epithelial cells. These findings suggest that epithelial cells play an important role in promoting ALPase expression in pulp fibroblastic cells. The co-culture system developed here will be useful for examining the role of the epithelial-mesenchymal interaction during odontoblast differentiation.