Osteosarcomas contain variable amounts of bony tissue, but the mechanism of bone formation by osteosarcoma is not well understood. While a number of cultured human osteosarcoma cell lines have been established, they are maintained by different media and differ qualitatively with regard to bone formation. We examined different media for their ability to support bone formation in vitro and found the alpha-modification of Eagle's minimal essential medium supplemented with beta glycerophosphate was best for this purpose, because it contained the proper calcium and phosphate concentrations. Subsequently, we compared seven human osteosarcoma cell lines under the same experimental conditions to clarify their ability to induce bone formation. NOS-1 cells most frequently exhibited features of bone formation in vitro and in nude mice. Collagen synthesis by tumour cells themselves seemed to be the most important factor for bone volume. However, even HuO9 cells, which lacked collagen synthesis and failed to form bone in vitro, successfully formed tumours containing bone in nude mice. Histological analysis of HuO9 cells in diffusion chambers implanted in nude mice and the findings of polymerase chain reaction indicated that the phenomenon was probably due to bone morphogenetic protein.