Factors that regulate growth and development of mesenchymal precursor cells towards chondrogenesis are not well identified. In the present study, we have developped a defined serum-free culture system which allows proliferation of chicken embryo tibia prechondrogenic cells (stage H.H. 28-30) and their subsequent maturation towards hypertrophic chondrocytes. Cell proliferation was obtained in adherent conditions in medium supplemented with insulin (Ins), thriiodothyronine (T3) and dexamethasone (Dex) at physiological concentration together with either bFGF, PDGFbb, EGF or GH at 1-10 ng/ml, bFGF presenting a more potent proliferative effect than the other growth factors. The stimulation promoted by the hormonal mixture and bFGF was synergistic as proliferation depended on their association. In particular the cycling cooperative effect occurred through Ins and/or Dex and not T3. Insulin could be substituted by IGF-I suggesting the involvement of the saturation of the IGF-I receptors. Prechondrogenic cells expanded in this defined medium, when transferred to suspension culture in T3/Ins/Dex as previously described (Quarto et al. J. Cell Biol., 1992), without growth factor supplement were able to undergo the complete chondrogenic development characterized by the inhibition of type I collagen synthesis, the sequential activation of type II and type X collagen synthesis and the concommitant morphological passage to cellular hypertrophy. In the differentiation step, insulin could not be substituted by IGF-I. In any condition the addition of BMP enhanced the differentiation process. Chondrogenesis was histologically evidenced by the formation of hypertrophic cartilage when the defined medium was supplemented with ascorbic acid. Assays in nude mice are presently under progress to assess whether cycling prechondrogenic cells maintained in the defined culture conditions are able to reproduce in vivo the endochondral cartilage maturation.