We have previously generated an immortalized human fetal osteoblastic cell line (hFOB) using stably transfected temperature sensitive SV40 T-antigen (Harris et al. [1995a] J. Bone. Miner. Res. 10:178-1860). To characterize these cells for phenotypic/genotypic attributes desired for a good cell model system, we performed karyotype analysis by multicolor fluorescent in situ hybridization (M-FISH), their ability to form bone in vivo without developing cell transformation, and finally their ability to form extracellular matrix formation in vitro. The karyotype analysis of hFOB cells revealed structural or numeric anomalies involving 1-2 chromosomes. In contrast, the human osteosarcoma MG63 cells displayed multiple, and often complex, numeric, and structural abnormalities. Subcutaneous injection of hFOB cells in the presence of Matrigel into nude mice resulted in bone formation after 2-3 weeks. Electron microscopic analysis of the extracellular matrix deposited by hFOB cells in culture revealed a parallel array of lightly banded fibrils typical of the fibrillar collagens such as type I and III. These results demonstrate that the hFOB cell line has minimal chromosome abnormalities, exhibit the matrix synthetic properties of differentiated osteoblasts, and are immortalized but non-transformed cell line. These hFOB cells thus appear to be an excellent model system for the study of osteoblast biology in vitro.