Transforming growth factor$ (TGFP) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone-forming cells. TGFP has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast-like osteosarcoma cell line ROS 1712.8. Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation. To test the hypothesis that TGFP plays a role in regulating mineral deposition in the matrix, the effects of TGFP on ALPase and phospholipase A, , two enzymes associated with mineralization, were examined. ROS 1712.8 cells were cultured at high and low density with recombinant human TGFP (0.1-10 ng/ml) to examine the influence of cell maturation on response to TGFP. Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high-density cultures, there was further stimulation at 10 ng/ml. There was a dose-dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures. Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme. However, in low-density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles. There was a dosedependent increase in phospholipase A,-specific activity in the plasma membranes and matrix vesicles of both high-and low-density cultures. In agreement with previous studies, TGFP inhibited cellular proliferation 50%. The results show that addition of TGFP stimulates the activity of enzymes associated with calcification. The effect of TGFP is dependent on the stage of maturation of the cell. This study indicates that TGFP may play an important role in induced bone formation, calcification, and fracture repair in addition to its role in promoting chon d rogenes i s.