Three clonal cell lines with differences in responsiveness to parathyroid hormone (PTH), alkaline phosphatase activity, and ability to produce an endothelial cell growth inhibitor(s) during more than 3 years, more than 58 passages, in culture were established from growth cartilage (GC) of mouse ribs. In sparse cultures the three clonal cell lines, MGC/T1.4, MGC/T1.17, and MGC/T1.18, all showed fibroblast-like morphology. However, as they became confluent, MGC/T1.4 cells became polygonal and then multilayered. MGC/T1.18 cells also became polygonal, but showed contact inhibition. MGC/T1.17 cells remained fibroblastic in confluent cultures and formed nodules when cultured for more than 7 days after they became confluent. These nodules calcified in the presence of beta-glycerophosphate. Glycosaminoglycan (GAG) synthesis in the parent uncloned line, MGC/T1 cells, at early passages was about 50-75% of that of primary cultures of mouse GC cells. The GAG syntheses in the three clonal lines were much lower than that of primary cultures of GC cells. Moreover, the sizes of proteoglycan monomers synthesized by these cells were not the same as that of cartilage-specific proteoglycan. The three clonal lines mainly synthesized type I collagen. PTH increased the intracellular cyclic AMP level in MGC/T1, MGC/T1.4, T1.17, and T1.18 cells: their maximal levels, observed after 2 minutes, were, respectively, about 160, 150, 70, and 200 times that of controls. The activity of alkaline phosphatase in MGC/T1.17 cells was higher than that in primary cultures of mouse GC cells, whereas those in MGC/T1 and T1.4 cells were comparable with that of GC cells, and that in MGC/T1.18 was lower. The three clonal lines, and especially MGC/T1.4, secreted a heat-stable, nondializable growth inhibitor(s) of endothelial cells into the culture medium. Because of their different properties, these cell lines should be useful for studies on endochondral ossification, the actions of PTH on skeletal cells, and anti-angiogenesis factors.