Dental pulp is a potential source of cells that can be used in cell replacement therapy for various nervous system disorders. Here we report that adult rat dental pulp cells have the ability to form neurospheres when cultured in serum-free culture medium on super-hydrophilic plates. The cells within small spheres continued to grow, and the dental pulp-derived cells generated large spheres. Sphere formation was dependent on exogenously supplied basic-fibroblast growth factor, but not on epidermal growth factor, and the formation and growth of dental pulp-derived spheres were negatively regulated by transforming growth factor-β. Plating cells that were dissociated from spheres on an adhesive substrate resulted in differentiation into Tuj1- and MAP2-positive neuronal cells. Analysis of the three-dimensional structure of dental pulp-derived spheres shows that they contained nestin-positive progenitors, Tuj1-positive neuronal cells and S100-positive glial cells. We found that spheres contained CD81 (TAPA1) and nestin double-positive cells, and identified a small population of CD81 and nestin double-positive cells in the odontoblast layer of the dental pulp. Flow cytometric analysis showed that CD81-positive cells were enriched in the spheres compared with the dental pulp tissue. Bromodeoxyuridine (BrdU) staining showed that nestin- and BrdU-positive cells were located only in the apical portion of the dental pulp, and the apical portion produced a large number of large-sized spheres. These data suggest that the CD81 and nestin double-positive cells localized in the odontoblast layer of the apical portion of the dental pulp may have the ability to grow and form neurospheres.