We have examined several analogs of 1α,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] in an animal model of osteoporosis (ovariectomized rats) to identify a compound with a greater therapeutic range than 1,25-(OH)₂D₃ for treatment of this bone disease. Here, we report that one analog, Ro-26-9228, had a bone-protecting effect but did not induce hypercalcemia at a wide concentration range. Analysis of biochemical markers and the bone histomorphometry of analog-treated rats suggested that Ro-26-9228 acted by inhibiting bone resorption and increasing the number of differentiated osteoblasts. To determine the basis for the segregation between hypercalcemia and bone-protecting action, we examined gene expression in tissues that regulate calcium homeostasis. We found that 1,25-(OH)₂D₃ induced 24-hydroxylase mRNA expression in the duodena of ovariectomized rats, but Ro-26-9228 did not. Furthermore, in the duodena of intact animals, 1,25-(OH)₂D₃ induced a significant increase in calbindin D 9K and plasma membrane calcium pump 1 mRNAs, but Ro-26-9228 had no effect on these mRNAs. On the other hand, the osteoblast-specific gene products osteocalcin and osteopontin were significantly up-regulated in trabecular bone by both the natural hormone and Ro-26-9228. Further investigation of gene-regulatory events in trabecular bone revealed that both 1,25-(OH)₂D₃ and Ro-26-9228 up-regulated TGF β1 and β2 mRNAs. We concluded that the unique properties of Ro-26-9228 include preferential gene regulation in osteoblasts over duodenum and effective induction of growth factors in bone.