Mesenchymal stem cells (MSCs) are self-renewing cells with the ability to differentiate into various mesodermal-derived tissues. Recently, we have identified in adult human periodontal ligament (PDL) a population of stem cells (PDL-MSCs) with the ability to differentiate into osteoblasts and adipocytes. The aim of the present work was to further characterize this population and the expression profile of its cells. To achieve our objective we have used flow cytometry, magnetic cell sorting, cytokine antibody array, and light and electron microscope immunostaining. Our results show that the PDL-MSCs contain a subpopulation of frizzled-9 (CD349) positive cells expressing a panel of key mesenchymal and embryonic markers including CD10, CD26, CD29, CD44, CD73, CD90, CD105, CD166, SSEA-1, and SSEA-4. They are additionally positive for nanog and Oct-4; two critical transcription factors directing self-renewal and pluripotency of embryonic stem cells, and they also express the cytokines EGF and IP-10. The presence of nanog, Oct-4, SSEA-1, and SSEA-4 suggests that PDL-MSCs are less differentiated than bone marrow-derived MSCs. Taken together, these data indicate the presence of immature MSCs in PDL and suggest that the frizzled-9/Wnt pathway plays an important role in regulating proliferation and differentiation of these cells.