This article describes experiments that were performed to examine the direct action of estrogen metabolites on cultured human osteoblast cells. The human fetal osteoblastic cell line, hFOB/ER9, which expresses high levels of the estrogen receptor (ER) alpha, was used to examine the direct effects of 16α-hydroxyestrone (16α-OHE1) and 2-hydroxyestrone (2-OHE1) on osteoblast differentiation. The 16α-OHE1 caused a decrease in osteocalcin (OC) secretion to a maximum of 40% of control values (vehicle-treated cells) at 10−7 M. Alkaline phosphatase (AP) activity was significantly induced at 10−7 M 16α-OHE1 with greater than 500% of control at 10−6 M 16α-OHE1. Finally, AP steady-state messenger RNA (mRNA) levels were increased within 24 h of 16α-OHE1 treatment. In contrast to 16α-OHE1, 2-OHE1 had no effects on the secretion of OC, AP activity, or AP gene expression. The 2-OHE1 also did not display any antiestrogen activity because treatment in combination with 17β-estradiol (E2) and 16α-OHE1 had no significant effect on the reduction in OC secretion or induction of AP activity. Similar to E2, 16α-OHE1 stimulated the expression of an early response gene, a TGF-β inducible early gene, designated TIEG, as early as 60 minutes after treatment, whereas treatment with 2-OHE1 displayed no effect. Support that the 16α-OHE1 regulation of these osteoblasts (OB) markers was mediated through the ER is shown by the fact that the estrogen antagonist ICI 182,780 abrogated these effects. These data suggest that 16α-OHE1 is a potent estrogen agonist on human osteoblastic hOB/ER9 cells. In contrast, 2-OHE1 displayed no estrogenic or antiestrogenic activity in this human osteoblast cell model.