In the development of secondary bone, mineralization of the cartilage matrix is the first step in endochondral mineralization. The circumstances of cartilage mineralization are not known. Influences of the periosteal tissue have been mentioned. In order to investigate the role of osteoblastic cells in endochondral mineralization, cartilage organoid cultures were induced to mineralize by the addition of beta-glycerophosphate (beta-GP). In cartilage organoid culture, embryonic mouse limb bud mesenchymal cells were grown at high-density. The cells differentiated into mature chondrocytes and produced hyaline cartilage matrix. When cartilage had formed after 6 days in vitro, 10 mM beta-GP was added. The developed mineralized cartilage was investigated by morphological means. Seven days after the addition of beta-GP, the first mineralized spots were visible mainly in the internodular, noncartilage tissue. After 12 to 14 days, large areas of cartilage were mineralized, and after 21 days, nearly the whole culture had been mineralized. Electron microscopic investigations showed a dramatic alteration of the cartilage matrix followed by a homogeneous mineralization of the cartilage matrix. The chondrocytes in the mineralized area died and faded. Typical rod-like apatite crystals were visible at the border between the mineralized and the unmineralized matrix. This result closely resembles the in vivo situation of cartilage mineralization. Addition of osteoblastic calvarial cells enhanced the mineralization process, as did the addition of conditioned medium of calvarial cell monolayers. Under these treatments, mineralization started after 3 days and reached a maximum after 14 days. On the other hand, addition of mouse skin fibroblast-like cells without a direct contact to the cartilage inhibited cartilage mineralization. These results indicate that osteoblastic cells induce endochondral mineralization, whereas fibroblast-like cells inhibit this mineralization via soluble factors.