Our long-term goal is to define cell populations and methods that can be used for autologous tooth tissue engineering in humans. The ability to identify and manipulate adult dental stem cells (DSCs) would significantly facilitate this process. Although a considerable body of literature supports the existence of epithelial and mesenchymal DSCs, DSCs themselves remain poorly characterized relative to other stem cells including neural, bone marrow stromal, hematopoetic, spermatogonial and keratinocyte stem cells. In particular, tools such as antibodies to DSC surface markers that could be used to generate enriched DSC populations, to identify adult DSC populations, and to examine the induction of odontogenic potential in non-dental tissues, are currently lacking. We hypothesize that the identification and characterization of post-natal dental stem cell (DSCs), combined with state-of the art tissue engineering techniques, will facilitate whole tooth tissue engineering efforts. Our recent tooth tissue engineering results demonstrate our ability to isolate, and maintain in culture, epithelial and mesenchymal post-natal DSCs, and to use these cells to bioengineer small tooth crowns containing dentin and enamel. Based on these promising initial results, here we propose studies whose successful completion will define methods to bioengineer teeth of predetermined size and shape. We propose: 1) to establish and characterize enriched epithelial and mesenchymal DSC populations using well-characterized stem cell sorting techniques, and to confirm their identity using our functional tooth tissue-engineering assay; and 2) to define scaffold materials and design for optimized bioengineering of tooth tissues of specified size and shape. The successful completion of the proposed aims will significantly improve our knowledge and understanding of DSCs and their potential utility in tooth tissue engineering, and will provide the foundation for eventual autologous tooth tissue engineering in humans.