1α,25-Dihydroxyvitamin D 3 [1α,25(OH) 2D 3] treatment of osteoblastic cells elicits a series of measurable responses that include both rapid, membrane-initiated effects and longer-term nuclear receptor-mediated effects. Structural analogs have been identified and characterized that selectively activate subsets of these pathways. Two analogs from over 35 that have been tested were chosen for this comparison because they activate non-overlapping response pathways, presumably representing either membrane-initiated or nuclear receptor-initiated activities. Compound AT [25(OH)-16ene-23yne-D 3] lacks the 1-hydroxyl essential for interacting with the nuclear receptor, but triggers Ca 2+ influx through plasma membrane Ca 2+ channels, augments parathyroid hormone (PTH)-induced Ca 2+ signals, dephosphorylates the matrix protein osteopontin (OPN), and along with PTH stimulates release of calcium from calvaria in organ culture. Compound BT [1α,24(OH) 2-22ene-24cyclopropyl-D 3] does not elicit any of the rapid responses or enhance PTH-induced bone resorption, but binds to the nuclear receptor for 1α,25(OH) 2D 3 and increases steady state mRNA levels of both OPN and osteocalcin over a 48 h period. Together, these two analogs recapitulate all of the known actions of 1α,25(OH) 2D 3 on osteoblasts. Based on these findings, we conclude that Ca 2+ release from bone stimulated by 1α,25(OH) 2D 3 and PTH is related to the rapid, membrane-initiated actions and is not likely to involve binding to the nuclear receptor for 1α,25(OH) 2D 3. Longer term stimulation of bone formation by 1α,25(OH) 2D 3, however, appears to involve solely the nuclear receptor-mediated effects. These findings support our model of 1α,25(OH) 2D 3 as a coupling factor for bone resorption and formation during bone remodeling.