1,25(OH)(2)D(3) enhances parathyroid hormone (PTH)-induced Ca(2+) signaling in osteoblasts by activating plasma membrane voltage-sensitive Ca(2+) channels (VSCCs). The ability of 1,25(OH)(2)D(3) or the VSCC-activating analog AT (25-hydroxy-16-ene-23-yne-D(3)) to enhance parathyroid hormone-stimulated (45)Ca(2+) release from cultured new-born rat calvaria was measured. Analog BT (1,24-dihydroxy-22-ene-24-cyclopropyl-D(3)), that does not mobilize Ca(2+), also was tested along with PTH. Control experiments were performed with and without PTH and with and without serum. Individual calvaria labeled in utero with (45)Ca(2+) were cultivated in serum-free medium on filters at the medium/air interface of 24-well culture plates and (45)Ca(2+) release followed over 72 h. The results demonstrated that 1,25(OH)(2)D(3) and the Ca(2+)-mobilizing analog, AT, but not the nuclear receptor-binding analog, BT, enhanced PTH-stimulated (45)Ca(2+) release under serum-free conditions. This enhancement effect of the seco-steroids was not evident in the presence of 10% fetal calf serum. The effect of analog AT was faster than that of 1,25(OH)(2)D(3). Nitrendipine, a specific L-type VSCC blocker, attenuated enhancement by vitamin D compounds, indicating that the high-threshold L-type VSCC is a molecular transducer of costimulation. These results emphasize the synergy between the calcitropic hormones 1,25(OH)(2)D(3) and PTH in cultures containing osteoblasts and osteoclasts, and suggest that the Ca(2+)-mobilizing activity of 1,25(OH)(2)D(3) enhances Ca(2+) release from bone.