We have found previously that the skeleton of adult female rats contains dexamethasone (Dex)- and progesterone (Prog)-dependent osteoprogenitors, and that estrogen treatment in vitro upregulates proliferation and differentiation of the Prog-dependent but not of the Dex-dependent osteoprogenitors (Bone 1997;20:17-25). The purpose of the present study was to determine whether ovariectomy (OVX) would have different effects on these two classes of osteoprogenitors. Six-month-old Sprague-Dawley rats underwent OVX and the lumbar vertebrae and proximal femurs were collected 1.5, 3, and 6 months after OVX. Cells were obtained from outgrowths of explant cultures and grown in α-MEM with 10% FBS, 50 μg/ml ascorbic acid, and 5 mM β-glycerophosphate. Osteoprogenitors were identified by their ability to generate a colony of osteoblastic cells forming bone (bone nodule). We also evaluated the number of colony-forming units - fibroblast (CFU-F) and of alkaline phosphatase (AP)-positive CFU-F. In cell populations obtained from vertebrae of rats ovariectomized for 1.5, 3, and 6 months and their corresponding control rats, both Dex (1-100 nM) and Prog (1-10 μM) dose-dependently stimulated nodule formation. Both Dex- and Prog-induced nodule formation were higher in cell populations from control rats than in those from ovariectomized rats (P < 0.001). Numbers of CFU-F and AP-positive CFU-F were also higher in cell populations from control rats compared with those from ovariectomized rats. Estrogen (10 nM) enhanced Prog-dependent bone nodule formation but decreased Dex-dependent bone nodule formation in populations from both control and ovariectomized rats. In femoral populations, the responses to Dex (10 nM), Prog (3 μM), and estrogen (10 nM) were similar to those of the vertebral populations in both control and ovariectomized rats. Our results demonstrate that ovariectomy in rats results in a dramatic decrease in the number of both Dex- and Prog-dependent osteoprogenitors in cell populations from vertebrae and proximal femurs. In addition, we confirmed our previous observation that estrogen upregulated proliferation and differentiation of Prog-dependent progenitors, but found here that estrogen clearly downregulated proliferation and differentiation of the Dex-dependent progenitors. © 2003 Elsevier Inc. All rights reserved.