The aim of this study was to compare transcriptional regulation in vivo during anabolic bone formation induced by either estradiol (E2) treatment or intermittent parathyroid hormone[1-34] (PTH) therapy. We utilized an ovariectomized (OVX) mouse model of osteoporosis and transcriptional profiling to identify genes upregulated by either high-dose E2 or PTH. Five weeks post-OVX, the mice were administered either E2 and/or PTH, or vehicle for 4 weeks. Femoral bones were analyzed by microCT and histomorphometry to confirm the anabolic effect of each treatment. OVX vehicle-treated control mice lost metaphyseal trabecular bone, with significant decrease in trabecular number, thickness, and connectivity. Both E2 and PTH treatments increased trabecular and cortical bone indices above the level of the sham operated controls, fully restoring both bone volume and bone mineral density (BMD). Moreover, PTH/E2 combination treatment led to significantly greater increase in cancellous bone and BMD than would be expected from the additive effects of the separate treatments. To determine whether PTH and E2 treatments were stimulating similar bone anabolic mechanisms, or were activating distinct signaling pathways, we compared patterns of gene expression using transcriptional profiling after either E2 or PTH treatment. After 4, 11, and 24 days of treatment, total RNA was collected from both the distal femoral metaphysis and diaphysis. Transcriptional profiling was performed using Affymetrix GeneChip probe arrays, comprised of approximately 36,000 full-length mouse genes and EST clusters from the UniGene database. Several markers of osteoblast activity, including c-fos, RANKL, PHEX, and PTHR1, were consistently upregulated by PTH in both skeletal sites. PTH treatment also increased expression of Cathespin K, consistent with the predicted increase in osteoclast activity. E2 treatment upregulated a largely distinct set of genes, including TGFβ3, and BMP1, as well as several genes critical for cell cycle control, including Cyclin D1 and CDK inhibitor 1A. Overall, comparison of transcriptional profiles suggest that anabolic responses in bone to PTH and high-dose E2 treatment after OVX-induced osteoporosis involve largely distinct patterns of gene regulation, each resulting in restoration of bone mass. © 2004 Wiley-Liss, Inc.