We find that the activity of a 0.4-kb human c-fos gene promoter (-404/+41), which lacks consensus estrogen-responsive elements (EREs), is regulated by estrogen receptor (ER) ligands in MC3T3-E1 osteoblastic cells through ERs in a manner distinct from ERE-mediated regulation. When ER alpha is coexpressed, both estrogens and antiestrogens upregulate promoter activity. When ER beta is coexpressed, however, three tested antiestrogens affect c-fos promoter activity, with tamoxifen exerting the greatest effect, while estrogens have no such effect. The tamoxifen agonism through ER beta is antagonized by 17 beta -estradiol, while the 17 beta -estradiol agonism through ER alpha is canceled by excess-level coexpression of ER beta. Deletion analysis revealed that the sequence -206/-110 plays a crucial role in the ER beta -mediated tamoxifen agonism. Interestingly, there is no ER beta -mediated tamoxifen agonism when nonosteoblastic cells are tested. Taken together, these results suggest that the transcription of the c-fos gene is regulated by ER ligands possibly through non-ERE elements in ligand structure-, cell type-, and ER subtype-dependent manners.