Chondrogenic promotion by rhGDF5 with or without rhTGFbeta3 was studied in pellet culture of human mesenchymal stem cells (HMSCs). A synergy between rhGDF5 and rhTGFbeta3 was observed in promoting chondrogenesis. rhBMP2, rhBMP6, rhBMP7 and rhTGFbeta1 were further tested and showed the same effect. To explore the mechanism, the expression of TGFbetatype I and II receptors, ALK5, ALK2, ALK3, ALK6, TGFbetaRII, BMPRII, ActRII was studied. ALK6 showed increase by the rhTGFbeta1 or rhTGFbeta3 treatment. ALK6 protein expression also showed increase by rhTGFbeta3. rhTGFbeta1/rhTGFbeta3 induced ALK6 up-regulation was inhibited by SD-208, a TGFbeta type I receptor inhibitor. Chondrogenesis by rhTGFbetal/rhTGFbeta3 or the combination between rhTGFbetal/rhTGFbeta3 and rhGDF5 also was diminished by SD-208. SMAD1/5/8 phosphorylation in nascent human mesenchymal stem cells (HMSCs) was stimulated weakly by rhGDF5 but strongly by rhBMP7. The rhGDF5 stimulated SMAD1/5/8 phosphorylation was enhanced by rhTGFbetal/rhTGFbeta3 but inhibited by SD-208. The rhBMP7 stimulated SMAD1/5/8 phosphorylation did not show influence by rhTGFbeta3 and SD-208. Our results indicated the potential involvement of ALK6 activation by rhTGFbetas in the synergy between rhTGFbetas and rhBMPs.