1, 25-dihydroxycholecalciferol (1, 25(OH)2D3) rapidly affects calcium (Ca2+) transport in several cell systems, suggesting physiological actions independent of genomic activation. To test this hypothesis, we studied immediate to early effects (0.5-300 sec) of 1, 25(OH)2D3on cytosolic Ca2+[Ca2+h in single osteogenic sarcoma ROS 17/2.8 cells loaded with fura-2. An acute rise in [Ca2+]iwas observed in 40% of the cells following addition of 1, 25(OH)2D3, with a threshold concentration of 10-11M. In most cases, the [Ca2+]irise was transient, with return to baseline within 1 min; less frequently a more prolonged effect was observed, with variable recovery times. 25-hydroxycholecalciferol (25(OH)D3) reproduced the effect of 1, 25(OH)2D3on [Ca2+]i(with equal potency and similar responses, whereas 24, 25- dihydroxycholecalciferol, la-hydroxycholecalciferol, and 22 oxa- 1, 25(OH)2D3were not effective. 1, 25(OH)2D3also increased [Ca2+]iin ROS 24/1 cells, which are defective of receptors for the vitamin D metabolites. At high doses (10-8-10-7M) of 1, 25(OH)2D3the [Ca2+]jrise in ROS 17/2.8 cells was due to both influx of extracellular Ca2+and release of Ca2+from intracellular stores, as the effect was only partially inhibited by Ca2+-channel blockade by nifedipine. At low doses (10-9-10-10M), the effect was entirely dependent on extracellular Ca2+. 1, 25(OH)2D3also increased the production of inositol 1, 4, 5 trisphosphate (Ins(l, 4, 5)P3) and diacylglycerol, at a threshold dose of 10-9M, indicating activation of phospholipase C (PLC). In two thirds of the cells studied, a second addition of 1, 25(OH)2D3within 5 min to cells prestimulated with equimolar doses of the vitamin D metabolite resulted in a [Ca2+]; transient of higher amplitude than the first, a phenomenon occurring at all doses of the hormone, and associated with production of Ins(l, 4, 5)P3. This response amplification was not produced by 25(OH)D3, and pretreatment with la(0H)D3did not significantly enhance l, 25(OH)2D3-induced production of Ins(l, 4, 5)P3. In conclusion, activation of the Ca2+message system by vitamin D metabolites is a rapid, nongenomic effect; 1, 25(OH)2D3specifically activates both PLC and dihydropyridine-sensitive Ca2+channels, and “primes” the cells to respond with an enhanced [Ca2+]irise to a subsequent homologous stimulation; the presence of both the la and 25 hydroxyl groups is necessary to express the full hormonal action of vitamin D on [Ca2+]i.