Osteoblast-like osteosarcoma cells (ROS 17/2.8) display a rapid transmembrane influx of extracellular calcium after stimulation by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] that is mediated largely by the opening of voltage-gated calcium channels. These cells also constitutively express high numbers (greater than 18,000/cell) of nuclear receptors for this seco-steroid hormone that are involved in the modulation of genomic activity in the osteoblast and in the up-regulation of transcript ion of osteoblast-specific genes such as osteocalcin. The objective of this study was to determine the structural hierarchy of vitamin D3 analogs with regard to their efficacy as molecular transducers of the genomic and nongenomic pathways that are activated upon treatment of osteoblasts with 1,25-(OH)2D3. To test the structural features of the agonist required for initiation of these distinct pathways, a series of ligand analogs and naturally occurring metabolites of 1,25-(OH)2D3 were used that contain A-ring, D-ring, and side-chain modifications. The abilities of these analogs/metabolites to 1) bind to nuclear receptors and 2) stimulate transmembrane calcium influx were measured. Several analogs (25-hydroxy-16-ene-23-yne-D3 and 25-hydroxy-23-yne D3) were found to stimulate Ca2+ channel opening, but bind only poorly to the 1,25-(OH)2D3 nuclear receptor. Conversely, other analogs (1,24-dihydroxy-22-ene-24-cyclopropyl D3 and 1,25-dihydroxy-16-ene-23-yne,26,27 F6-D3) were found to bind very well to the nuclear receptor, but displayed little or no activity in opening Ca2+ channels. Pertussis toxin, which interferes with coupling of certain ligand-gated receptors to ion channels, failed to block the activation of calcium channels by 1,25-(OH)2D3 or active agonist analogs. Our results indicate that there are likely to be distinct nuclear and plasma membrane-associated forms of the 1,25-(OH)2D3 receptor that are involved in genomic and nongenomic activation of osteoblast activity, respectively. The membrane-associated receptors do not appear to be coupled to pertussis toxin-sensitive G-proteins.