OBJECTIVE:Semaphorins are a group of secreted and membrane-associated molecules that play important roles in axon navigation. Several semaphorin family molecules are expressed in the pharyngeal arches and tooth germs. We analysed the expression of membrane-associated Semaphorin 4D (Sema4D) in tooth germs, and examined its potential role in regulating the differentiation of rat incisor pulp-derived cells in vitro. DESIGN:mRNA expression was examined by in situ hybridisation. The effects of Sema4D on rat pulp-derived cells were examined by adenovirus-mediated overexpression in vitro. RESULTS:Both epithelial and mesenchymal cells of tooth germs expressed Sema4D at the early bell stage. Later, the odontoblasts predominantly expressed Sema4D, while the epithelial expression greatly decreased. The overexpression of Sema4D in rat incisor pulp-derived cells strongly inhibited mineralisation. This inhibition was preceded by a reduction of collagen fibre production at the level of mRNA synthesis. CONCLUSIONS:These results indicate that Sema4D is expressed in both epithelial and mesenchymal cells of the tooth germs. Sema4D represses collagen synthesis of pulp-derived cells, indicating it might negatively regulate odontoblast differentiation.