The molecular mechanisms that control the function of periodontal ligament (PDL) fibroblasts remain unclear. We speculated that the character of differentiating PBL fibroblasts is defined by the altered expansion of specific genes not found in neighboring gingival fibroblasts in the periodontium. To expand this set, subtractive hybridization was applied between cultured human PDL and gingival fibroblasts to identify genes differentially ex-pressed in PDL. Consequently five candidate clones, PDLs (periodontal ligament specific)5, -17, -22, -25, and -31 were identified and characterized by homology search, Northern analysis, and irt situ hybridization. Although the mRNAs of these clones were expressed by bone marrow cells and rarely by gingival fibroblasts, the highest expression was detected in the PDL cells, which were uniformly distributed throughout the whole PDL. Amongst the five candidate clones, me focused on PDLs17, because it is a hypothetical protein whose biological function has not been reported Set in the database. Polyclonal antiserum raised against PDLs17 peptide was made, and stained the PDL fibroblasts, osteoblast-liIie cells and stromal cells in the bone marrow, but not gingival fibroblasts. The results suggest that clones, PDLs5, -17, -22, -25, and -31 may be used as PDL fibroblast-speeific markers, and that PDLs17 could act as an important factor in the differentiation process of PDL fibroblasts.