Dexamethasone increased alkaline phosphatase levels up to 7-fold in the osteoblast-like rat osteosarcoma cell line ROS 17/2.8. This effect was associated with reduced cell growth and took place over several days in culture. The increase in enzyme activity was dose dependent, (half-maximum near 1 nM, with a hormone specificity suggesting glucocorticoid receptor mediation). Dexamethasone also increased enzyme activity in ROS 2/3 cells, but not in two nonosteoblastic osteosarcoma cell lines, indicating that among these cell lines, the effect is specific for osteoblast-like cells. Moreover, enzyme activity in both control and dexamethasone-treated cells correlated directly with levels of radioimmunoassayable bone-type isoenzyme. Increases in alkaline phosphatase activity in response to dexamethasone were detectable after about 5 h and were inhibited by both actinomycin D and cycloheximide. Thus glucocorticoids appear to increase de novo enzyme synthesis in ROS 17/2.8 cells. Finally, the cAMP-elevating agents PTH, isoproterenol, and 8-bromo-cAMP, which were previously shown to reduce alkaline phosphatase activity in osteoblast-like cells, antagonized the effects of dexamethasone. Moreover, in the presence of dexamethasone, lower concentrations of these agents were required for inhibitory effects on alkaline phosphatase.