Cell lines were established from three spontaneous osteosarcoma and one fibrosarcoma of aging mice. They were studied for tumorigenicity, osteoblastic features, and other in vitro cellular characteristics, by a combination of histological, morphological, biochemical, and molecular approaches. It was found that all cell lines formed tumors in vivo, whereas in vitro, only the fibrosarcoma-derived cell line grew efficiently in soft agar. Three out of the four cell lines produced mouse endogenous retroviruses, but none were classical sarcoma viruses. Type I collagen was expressed by all the cell lines, as was another extracellular matrix protein, osteonectin. The osteosarcoma-derived cell lines, however, exhibited different degrees of osteogenic differentiation. Only one line (OSA), and its clonal subline (1G11), consistently gave rise to mineralized tumors after transplantation into syngeneic mice, and these cells expressed high levels of alkaline phosphatase and bone-specific osteocalcin mRNA in vitro. Expression of these biochemical markers of osteoblasts occurred to a lesser extent in a second line (OSC) and was undetectable in the third line (OSB). The clonal 1G11 cell line exhibits the phenotype of a fully mature osteoblast and thus may serve as a particularly useful model for studies of bone cell function and regulation. Studies of cells which display a wide spectrum of osteogenic potential may further our understanding of the mechanisms involved in bone cell differentiation and tumorigenicity.