Notes: Transforming growth factor-betas (TGF-βs) and bone morphogenetic proteins (BMPs), members of a TGF-β superfamily, are known to play an important role in osteogenic cell differentiation and consequently bone formation. We have reported previously that periodontal ligament (PDL) cells differentiate and form mineralized nodules when cultured in the presence of dexamethasone (Dex), β-glycerophosphate (GP) and ascorbic acid (AA). To understand the roles of TGF-β isoforms (TGF-β1, 2 and 3) and TGF-β type 1 receptors (activin receptor-like kinase (ALK)-2, -3, -5 and -6) in PDL cell differentiation, their expression was investigated using Northern blot analysis. Rat PDL cells, derived from coagulum in the tooth socket, were cultured in the presence of Dex (5 μM), GP (10 mM) and AA (50 μg/ml) for up to 21 d. Total RNA was isolated from PDL cells after 0, 7, 14 and 21 d and used for Northern blot analysis of mRNAs for matrix proteins, TGF-β isoforms and their receptors using 32P-labeled cDNAs as probes. Four stages showing distinct morphological characteristics and matrix expression during development of mineralized nodules were identified. Type I collagen (Col I) and SPARC (secreted protein. acidic and rich in cysleine) mRNAs were expressed at the confluent stage, but decreased during the mineralization stage. Osteopontin (OPN) and alkaline phosphatase (ALP) transcripts were initially observed at multilayer stage, while bone sialoprotein (BSP) and osteocalcin (OC) at the nodule stage and all 4 were expressed thereafter. TGF-β1 mRNA expression increased with the progression of PDL cell differentiation, while a relatively high level of TGF-β3 transcript decreased slightly during their differentiation. TGF-β2 mRNA was not expressed. The expression of TGFβ-RI mRNA decreased, whereas that of TGFβ-RIII increased dramatically with PDL cell differentiation. TGFβ-RII gene activities remained high throughout all stages. ALK-2, ALK-3 and ALK-6 mRNA expression increased with the progression of PDL cell differentiation, suggesting that these receptors may play important roles in Dex-induced PDL cell differentiation and mineralized nodule formation.