The estrogen-related receptor alpha gene encodes a nuclear receptor protein, ERR alpha, whose structure is closely related to the estrogen receptors. ERR alpha modulates estrogen receptor (ER)-mediated signaling pathways both positively and negatively. It is selectively expressed in a variety of cell types during development and in adult tissues. We have previously shown that estrogen stimulates the expression of the ERR alpha gene in mouse uterus. In this study, we found that the ERR alpha gene is stimulated by estrogen in mouse uterus and heart but not in liver. Estrogen also stimulates the expression of ERR alpha in the human breast and endometrial cell lines. The human ERR alpha gene promoter contains multiple Sp1 binding sites, and the Sp1 protein is required for the promoter activity. The major estrogen response is mediated by a 34-bp DNA element that contains multiple steroid hormone response element half-sites (MHREs) that are conserved between the human and mouse ERR alpha gene promoters. Mutations made at a single or multiple sites of the MHREs abolished the ER-mediated transcription of the element in transient transfection experiments. By chromatin immunoprecipitation assay, we demonstrated the interaction between ER alpha and MHREs of the endogenous ERR alpha gene promoter in MCF-7 cells. Estrogen treatment further enhanced the association of ER alpha and MHREs in vivo. The present study demonstrated that the ERR alpha gene is a downstream target of ER alpha.