Adult multipotent stem cells have been isolated from various non-neural tissues. Here, we report the isolation of multipotent stem cells from rat periodontal ligament (PDL) using neurosphere-forming culture system. Enzymatically dissociated PDL cells were cultured in serum-free basal medium containing EGF, bFGF, and LIF. Free-floating spheres expressing nestin, GFAP, and vimentin were formed by 7 days of the culture. In addition, spheres expressed mRNA of neural crest-associated transcription factors Twist, Slug, Sox2, and Sox9. PDL-derived spheres differentiated into multinucleated myotubes, NFM-positive neuron-like cells, GFAP-positive astrocyte-like cells and CNPase-positive oligodendrocyte-like cells. Methylcellulose colony-forming assay revealed that a single PDL cell could form a sphere at a frequency of approximately 0.01% of total cells. These data indicate that PDL-derived spheres contained multipotent adult stem cells capable of differentiating into both neural and mesodermal progeny. This is the first report of the isolation of PDL-derived stem cells with primitive neural crest stem cell features.