Extracellular inorganic pyrophosphate (PPi) is involved in the regulation of mineralization, and there is evidence that the cell surface enzyme, NTP pyrophosphatase, is a major source of this metabolite in bone. Osteotrophic agents that influence bone turnover may exert their effects, in part, by modulating the activity of ecto-NTP pyrophosphatase in bone cells. We investigated the effect of 1,25(OH)2D3,24,25(OH)2D3, dexamethasone, and parathyroid hormone (PTH) on the activity of this enzyme in cultured human trabecular bone-derived osteoblast-like cells. 1,25(OH)2D3 at 10−11-10−9 M induced a dose- and time-dependent increase in activity (at 96 h; maximum 10−9 M, p > 0.001), whereas higher concentrations (10−8 and 10−7 M) had no effect. In contrast, 24,25(OH)2D3 was effective only at 10−8 and 10−6 M (at 96 h; p > 0.01). Dexamethasone (10−9-10−7 M) caused a dose-dependent decrease in ecto-NTP pyrophosphatase activity (10−7 M, p > 0.001); concentrations higher than 10−7 M did not evoke greater inhibition. This effect became apparent by 48 h and was significantly enhanced after 72 h. The response to dexamethasone was attenuated by cycloheximide, indicating a requirement for de novo protein synthesis. Interestingly, the stimulatory effect of 10−9 M 1,25(OH)2D3 on ecto-NTP pyrophosphatase activity was significantly enhanced in the presence of dexamethasone (10−9-10−7 M). Human PTH(1-34) and bovine PTH(1-34) in the range 10−10-10−7 M had no effect on enzyme activity over a 72 h period. The effects of vitamin D3 on the expression of bone ecto-NTP pyrophosphatase may be tissue or cell type specific because the ecto-NTP pyrophosphatase activity of subject-matched skin-derived fibroblasts showed no sensitivity to 1,25(OH)2D3. These data suggest a possible role for both vitamin D3 metabolites and glucocorticoids in the regulation of the mineralization process in vivo via modulation of PPi production.