Muscle-derived cells can differentiate into chondrogenic cells. In our present study, we investigated the pattern of expression of Sox9, a transcription factor known to play a key role in chondrogenesis, in a rat myoblastic cell line, L6. In addition, we evaluated expression of type II collagen and myogenic regulatory markers by reverse-transcript polymerase chain reaction. We also investigated the effect of transforming growth factor (TGF)-beta3, which is known to induce chondrogenesis, on Sox9 mRNA expression. On the first day of culture, we observed a high expression of Sox9. However, on the seventh day of culture, there was a decline in the level of Sox9 and type II collagen mRNAs and an increased expression of Myf5 and myogenin mRNAs. Sox9 mRNA expression was increased after stimulation of TGF-beta3 at 2, 6, and 24 hr. Cartilage nodules were observed in L6 cells treated with TGF-beta3 and dexamethasone. These results indicated that L6 myoblasts originally possess the capacity to differentiate into chondrogenic cells, but that capacity is lost as the cells differentiate toward the myogenic lineage. In addition, TGF-beta3 may modulate Sox9 mRNA expression in L6 cells and retain the capacity to differentiate into chondrogenic lineage.