Transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) induce a cartilage-specific extracellular matrix (ECM) gene, aggrecan, in a chondrogenic cell line, ATDC5. The results of our recent study show that TGF-β1, but not BMP-4, strongly induces an ECM gene, fibronectin, during chondrogenesis. However, the role of fibronectin in chondrogenesis is unclear. In the current study, our results showed that TGF-β1, but not BMP-4, led to versican-dominant proteoglycan production during chondrogenesis of ATDC5 cells. siRNA-mediated reduction of fibronectin and interference in the liaison between fibronectin and integrins by the Arg-Gly-Asp-Ser (RGDS) peptide increased aggrecan expression, and decreased versican expression by TGF-β1 stimulation. These data suggest that fibronectin is a critical mediator for TGF-β-specific production balance of 2 major proteoglycans, aggrecan and versican, during chondrogenesis.