Recent characterization of human dental pulp stem cells has shed new light on the understanding of the odontoblastic lineage. The purpose of the study was to characterize human adult dental pulp cells isolated and cultured in vitro and to examine the cell differentiation potential grown on dentin. We observed that some pulp cells isolated with an enzyme-digestion approach proliferated at a similar rate as the immortal cell line NIH 3T3. Population doubling time (PDt) for pulp cells at passage 3 was 22.6 +/- 0.5 hours and for NIH 3T3 was 23.1 +/- 2.3 hours. The pulp cells formed mineral nodules stimulated with dexamethasone or dexamethasone plus 1,25-dihydroxyvitamin D3. Pulp cells, after being seeded onto mechanically and chemically treated dentin surface, appeared to establish an odontoblast-like morphology with a cytoplasmic process extending into a dentinal tubule revealed by scanning electron microscopy analysis. Our data demonstrated the formation of cells with odontoblastic morphologies on existing dentin, suggesting that isolated human pulp stem cells may differentiate into odontoblasts on dentin in vitro.