Neurotrophic factors have robust effects on development, differentiation, maintenance and regeneration of neurons. In the present study, we have used in situ hybridization to determine the specific sites of gene activity of five neurotrophic factors during tooth development. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNAs were mainly detected in the dental papilla/pulp in postnatal rats, and the pattern of expression correlated with onset of dental innervation. In contrast, neurotrophin 3 (NT3) and 4 (NT4) mRNA expression patterns were predominantly epithelial and were strongest during early developmental stages when teeth are not yet innervated. Glial cell line-derived neurotrophic factor (GDNF) mRNA was present in dental epithelium at early stages, but later in development, GDNF mRNA expression was mainly mesenchymal and observed in the odontoblast layer and extending into the subodontoblast zone. Our results suggest that both neurotrophins and GDNF may have multiple functions during tooth development. In addition to an influence on the establishment of the dental innervation, neurotrophic substances might have morphogenetic effects such as modulating the proliferation or differentiation of developing epithelial and mesenchymal cells.