['Although enamel matrix derivative can promote chondrogenic differentiation of pluripotent mesenchymal precursor cells, the molecular mechanism that underlies this phenomenon is unclear. The purpose of this study was to determine the effect of enamel matrix derivative on chondrogenic differentiation. ATDC5 cells, which undergo a reproducible multistep chondrogenic differentiation, were cultured with or without enamel matrix derivative for up to 35 d.', 'Cell proliferation and alkaline phosphatase activity increased markedly in cells cultured in the presence of enamel matrix derivative, compared with cells cultured in its absence. Deposition of Alcian blue-positive cartilage matrix and Alizarin red-positive mineralized nodules also increased significantly upon treatment with enamel matrix derivative. Expression of mRNAs encoding cartilage extracellular matrix proteins (type II collagen, type X collagen and aggrecan) and chondrogenic-related transcription factors (Sox9, Zfp60 and AJ18) were measured using the real-time polymerase chain reaction. Type II collagen, type X collagen and aggrecan mRNA expression increased markedly with enamel matrix derivative treatment. Transcription of Sox9, an important transcription factor that mediates chondrogenic differentiation, also increased with enamel matrix derivative treatment. The KRAB/C2H2 zinc-finger transcription factors, Zfp60 and AJ18, were transiently expressed in the prehypertrophic stage, and their expression increased with enamel matrix derivative treatment. In a western blot analysis with anti-insulin-like growth factor-I and anti-bone morphogenetic protein-6 immunoglobulin, bands corresponding to approximately 14, approximately 18 and approximately 60 kDa were found in enamel matrix derivative.'] Does enamel matrix derivative stimulate chondrogenic differentiation of ATDC5 cells?