Cells derived from the periodontal ligament (PDL) have previously been reported to have stem cell-like characteristics and to play an important part in re-building damaged tissue, including alveolar bone. However, these populations have been heterogeneous, and thus far no highly purified periodontal stem cell (PSC) clone has yet been established from adult human PDL tissue. The present study was therefore carried out to isolate single cell-derived PDL clones and to delineate their phenotypic and functional characteristics. In this report we have obtained four homogeneous and distinct clones--namely, C5, C6, C7, and C8--and have found these to be highly proliferative and to express the stromal cell markers CD29 and CD44. In particular, C7 showed stem cell-like characteristics of small cell size with reduced cytoplasm, clonogenicity, and multilineage potential, including osteogenic activity in forming bone-like tissue in organoid micromass cultures. Clones C5 and C6 possessed osteoprogenitor features with mineralized matrix-forming activity, whereas C8 did not undergo osteogenic, adipogenic, or chondrogenic differentiation. The present study thus reports, for the first time, the isolation and cellular and molecular characterization of highly purified putative PSC and osteoprogenitors in adult human PDL, based on clonogenicity and multilineage differentiation potential, with PSC-C7 capable of bone formation in vitro, suggesting that such cells may have potential value for stem cell-based bone tissue engineering in vivo.