Several clonal cell lines from a transplantable rat osteosarcoma, selected on the basis of parathyroid hormone (PTH)-sensitive adenylate cyclase, were established in culture. Bovine PTH-(1-84) (0.1 microM) stimulation of adenylate cyclase varied among clones from 8-fold to none. The level of PTH response was a stable property of each clonal line that was retained through numerous passages in vitro (nearly 3 yr in the oldest clone). Highly PTH-responsive lines had a cuboidal-eliptoid morphology and differed from the nonresponsive lines, which had a more fibroblastic appearance. PTH responsiveness correlated with several properties, presumably associated with the osteoblastic phenotype: elevated alkaline phosphatase activity, synthesis of the gamma-carboxyglutamic acid-containing bone protein, and production of mineralized tumors in host rats. PTH (1.0 nM; 24 h) reduced the alkaline phosphatase activity by 40% when tested in a responsive clone. The acid phosphatase activity of the various cell lines was uniformly low. These osteosarcoma-derived cell lines which are stable in vitro thus seem to reflect the phenotypic heterogeneity observed in the tumor in situ. They could be useful in studies of phenotypic expression, PTH action, and a possible relationship between the two.