The identity of the paracrine mediator(s) of the antiresorptive action of estrogen on bone cells is controversial. Osteoprotegerin (OPG) was recently identified as a soluble member of the tumor necrosis factor (TNF) receptor (TNF-R) superfamily that is secreted by osteoblast lineage cells and acts by binding to and neutralizing its cognate ligand, OPG-L, a required factor for osteoclastogenesis. OPG prevents bone loss when administered to ovariectomized rats, induces osteoporosis when ablated in knock-out mice, and induces osteopetrosis when overexpressed in transgenic mice. In conditionally immortalized, human osteoblastic hFOB/ER-3 and hFOB/ER-9 cell lines containing physiological concentrations of approximately 800 and approximately 8,000 functional estrogen receptors (ER)/nucleus, respectively, we found that 17beta-estradiol dose- and time-dependently increased OPG mRNA and protein levels to maximal levels of 370% and 320%, respectively (P < 0.001); co-treatment with the "pure" antiestrogen ICI 182,780 abrogated these effects completely. 17beta-Estradiol also dose-dependently increased OPG mRNA and protein levels in normal human osteoblasts with approximately 400 ER/nucleus by 60% and 73%, respectively. Thus, estrogen enhancement of OPG secretion by osteoblastic cells may play a major role in the antiresorptive action of estrogen on bone.