Regulated splicing of fibronectin (FN) occurs during the mesenchymal to chondrocyte transition and ultimately results in the relative enrichment of an extra domain B (EDB) exon-containing FN isoform with the suggestion that FN isoforms may play a functional role in chondrogenesis. Promotion of chondrogenesis can also be achieved by treatment with transforming growth factor-β (TGF-β), which also regulates FN isoform expression. We have examined the effects of TGF-β treatment on the assumption of the chondrogenic phenotype in the teratoma-derived cell line ATDC5 and tested whether these effects on chondrogenesis are paralleled by appropriate changes in FN isoform expression. ATDC5 cells were maintained in a pre-chondrogenic state and, in this state, treated with 10 ng/ml TGF-β. The cells started to elaborate a matrix rich insulfated proteoglycans, such that within the first 12 days of culture, TGF-β1 treatment appeared to slightly accelerate early acquisition of an Alcian blue-stained matrix, and caused a dose- and time-dependent decrease in collagen type I expression; changes in collagen type II expression were variable. At later times, cells treated with TGF-β became indistinguishable from those of the controls. Interestingly, TGF-β treatment caused a significant dose- and time-dependent decrease in the proportion of FN containing the extra domain A (EDA) and the EDB exons. These data suggest that TGF-β induces the early stages of chondrogenic maturation in this pre-chondrogenic line and that TGF-β treatment increases expression of FN isoforms that lack the EDA and EDB exons.