Specialized connective tissues such as tendon/ligament develop through a series of events that require temporal and spatial expression of numerous genes in mesenchymal progenitors. However, the genes required for tendon/ligament development have not been identified yet. To solve this problem, we made a cDNA library from periodontal ligament and sequenced 11,520 cDNA clones, as a model for investigating tendon/ligament development. The resulting sequence data was assembled to 617 expressed sequence tag (EST) clusters, and an EST database for human periodontal ligament (PDL) was constructed (designated as the KK-Periome database). In the KK-Periome database, the top 13 EST clusters were related to extracellular matrix (ECM) genes. The temporal and spatial expression patterns of these genes during mouse PDL development were examined by in situ hybridization. Among these genes, F-spondin was expressed specifically in dental follicle (DF) cells during tooth germ development, whereas tenascin-N was strongly expressed in the terminally differentiated PDL. This characteristic expression profile was confirmed by in vivo differentiation assay of human PDL (hPDL) cells in the mouse transplant. Thus, the KK-Periome database was proven to be a useful resource for PDL-derived ESTs (transcriptome), and in fact, initial evidence indicated that F-spondin and tenascin-N might serve as markers for DF and PDL, respectively.