We previously showed that retinoic acid (RA) participates in the regulation of chondrocyte maturation during endochondral ossification, a process involving multiple developmental stages. To assess whether the responsiveness to RA treatment changes during chondrocyte maturation, immature chondrocytes were isolated from the caudal portion of Day 18-19 chick embryo sterna, a portion that remains cartilaginous through early postnatal life but ossifies with age. The immature cells were allowed to reach different stages of maturation by growth for different time in culture. Progression by the cells toward the mature phenotype during culture was confirmed by increases in average cell diameter, proteoglycan synthesis, and alkaline phosphatase (APase) activity. When developmentally immature passage 0 (PO) cultures were treated with RA (10-100 nM) for 72 h, the cells readily became fibroblastic, reduced drastically their proteoglycan synthesis, and failed to activate type X collagen gene expression. When older cultures (P1 and P2) were treated with RA, the cells acquired a characteristic epithelioid shape and increased their APase activity. Moreover, 5-10% of P1 cells and 20-25% of P2 cells activated type X collagen synthesis in response to RA. RA treatment markedly induced expression of the gene encoding the beta isoform of retinoic acid receptor (RAR beta) and also provoked a moderate 2.5-fold increase in RAR alpha gene expression. A similar change in responsiveness to RA was observed during maturation in vivo. Chondrocytes were isolated from the cephalic portion of Day 10, 11, 13, and 16 chick embryo sterna, and were treated with different doses of RA (10-100 nM) for 72 h. The cells from the Day 10 sternum failed to activate type X collagen gene expression in response to RA. In contrast, with increasing age of the embryos, an increasing fraction of cells induced type X collagen gene expression in response to RA. We conclude that responsiveness to RA changes during the early stages of chondrocyte maturation and that maturation depends on interactions between exogenous retinoids and the endogenous developmental program of chondrocytes.