In UMR 106 rat osteosarcoma cells, parathormone (1-34hPTH) and calcitonin (sCT) stimulated adenylate cyclase (AC) activity 5.5-and 2.8-fold, respectively. AC in osteoblasts (OB) from collagenase-treated calvaria of 3-day-old rats responded similarly to 1-34hPTH. In contrast, fibroblasts (mouse fibroblastomas) displayed a marginal 1-34hPTH sensitive AC. Osteoclasts (OC) of collagenase-treated rat calvariae, rat monocytes and mouse macrophages did not demonstrate 1-34hPTH inducable AC activity. Physiological concentrations of 24,25-dihydroxyvitamin D-3 attenuated PTH-sensitive AC in OB and UMR 106 cells within 20 min, while 1,25-dihydroxyvitamin D-3 showed no such immediate effect. In contrast, the AC response to Gpp(NH)p was unaffected by 24,25-(OH)2D3, indicating that 24,25-(OH)2D3 interrupts the coupling of the PTH receptor to the GTP binding protein Gs. OB and UMR 106 cells were also subjected to long-term (48 h) incubation with vitamin D-3 metabolites, 1-34hPTH or 20% serum from patients with secondary hyperparathyroidism (sHBT-serum), respectively. PTH-sensitive AC was markedly attenuated by pre-exposure to both 1-34hPTH and 1,25-(OH)2D3, while minimally affected by corresponding 24,25-(OH)2D3 and 20% sHPT-serum treatment. The secretion of alkaline phosphatase (Alphos) from the two cell types was strongly increased by 1-34hPTH, the effect being abolished by the presence of 24,25-(OH)2D3. Iliac crest biopsies of normal individuals exhibited a clear negative correlation between PTH-sensitive AC and corresponding serum 24,25-(OH)2D3 levels. Basal AC activity was, however, negatively correlated to serum 1,25-(OH)2D3 concentrations. In summary, the results show that 24,25-(OH)2D3 reduces PTH-stimulated AC activity in and Alphos secretion from osteoblastic bone cells by rapidly and directly interfering with the plasma membrane. These data reinforce the probable in vivo significance of 24,25-(OH)2D3. Moreover, the negative correlation between basal AC activity and serum 1,25-(OH)2D3 levels indicates a possible role for 1,25-(OH)2D3 in regulating bone cell synthesis of AC components in vivo.