Objectives: To characterize isolated clonal post-natal epithelial and mesenchymal dental stem cells (DSCs). Methods: Molar tooth buds were isolated from 4 day post-natal rat pups, were used to generate enriched dental epithelial and mesenchymal cell populations. Single cell suspensions of isolated dental epithelial and mesenchymal cells were grown in vitro for 5 days, and subsequently sorted either by Hoechst dye exclusion to identify putative DSC side populations (SPs), or by magnetic bead immuno-sorting using the stem cell antibody anti-STRO-1. Sorted cell populations were subsequently tested in our established functional bioengineered tooth assay as described (Young et al., 2002; Duailibi et al., 2004). To characterize the transcriptional profile of quiescent, proliferating and differentiating dental stem cells and daughter cells, homogeneous clonal cell populations were generated. Clonal sorted cell populations are being generated and expanded in vitro. Expanded DSC and non-DSC lines are being examined, using both in vitro and in vivo methods, to define their differentiative potential. Results: To date, expanded 31 pulp organ (PO) SP clones, 44 PO non-SP clones, 90 enamel organ (EO) SP clones, and 66 EO non-SP clones have been generated. We are currently defining the transcriptional and differentiation profiles of individual and paired epithelial/mesenchymal cell lines. Conclusions: Clonal putative DSCs isolated from 4 dpf rat molar teeth exhibit distinct morphologies. Molecular and differentiation profiles will provide important characterizations of tooth bud cells, which will ultimately facilitate ongoing tooth tissue engineering efforts. Supported by: The Forsyth Institute, NIH/NIDCR grants R41 DE015445, R01 DE016132, and Dentigenix/Ivoclar-Vivadent.