A novel serum-free culture system was developed in an attempt to generate a three-dimensional hyalinelike neocartilage independent of polymer scaffolds. Neocartilage disks as much as 1.5 mm thick were produced, which were characterized by synthesis of the normal articular cartilage collagens and proteoglycans. In contrast to growth in serum-containing media, chondrocytes from juveniles maintained in static culture under defined serum-free conditions deposited an extracellular matrix that accumulated in the form of tissue disks. Electron microscopic evaluation of neocartilage disks revealed collagenous matrices characteristic of articular cartilage from human infants. The neocartilage did not show terminal chondrocyte differentiation as shown by the absence of Type X collagen production and lack of cellular hypertrophy. Although chondrocytes from preadolescent donor cartilage recapitulated embryonic development in the absence of exogenous factors, chondrocytes from articular cartilage from adults failed to produce neocartilage when grown under identical conditions. This is the first demonstration that autocrine morphogens are sufficient to guide production of hyaline cartilage in vitro. In addition to providing a unique model system to compare the healing response of mature and immature articular chondrocytes, this technology may be of clinical importance in the development of new biomaterials for repair of articular cartilage defects.