Previous studies have shown that 1,25(OH)2D3 activates multiple signaling pathways in osteoblasts, including rapid nongenomic and long-term genomic pathways. Genomic pathways are mediated by the vitamin D receptor (VDR), a member of the steroid receptor superfamily, and involve transcriptional regulation of target genes. Nongenomic pathways involve lipid turnover, activation of Ca+2 channels and elevation of intracellular Ca+2, all of which occur within seconds after addition of seco-steroid. The interaction of other physiological metabolites of vitamin D, such as 24,25(OH)2D3, with target cells such as osteoblasts is much less clear. We have used a combination of electrophysiological, biochemical, molecular and ion tracer studies to dissect the physiological responses of osteoblast-like osteosarcoma cells (ROS 17/2.8) to 1,25(OH)2D3 and related metabolites. We conclude the following: 1) the structural requirements for activation of genomic vs. nongenomic pathways by seco-steroid are distinct and likely to involve separate receptors; 2) activation of rapid nongenomic pathways is independent of the long-term regulation of target genes; and 3) 1,25(OH)2D3 and 24,25(OH)2D3 interact at the level of the plasma membrane to regulate Ca+2 permeability. Present studies are aimed toward the understanding of the role of both genomic and nongenomic pathways in osteoblast physiology.