The human estrogen-related receptor alpha 1 (hERR alpha 1) is an orphan member of the steroid/thyroid hormone receptor superfamily. A cDNA encoding this protein was originally isolated on the basis of sequence similarity in its DNA-binding domain with estrogen receptor alpha (ER alpha). Previously, we reported the purification of hERR alpha 1 from HeLa cell nuclear extracts on the basis of its ability to bind two sites in the late promoter of simian virus 40 (SV40). We have now determined the primary structure and the DNA and protein binding specificities of hERR alpha 1 and developed in vivo and in vitro assays for its functional activities. hERR alpha 1 was found to bind as a monomer, with a high-affinity binding site containing the extended half-site sequence 5'-TCAAG-GTCA-3'. Binding sites for hERR alpha 1 were identified in many cellular promoters, including some that were previously shown to function as estrogen-response elements (EREs). hERR alpha 1 was shown to function as a sequence-specific repressor of the SV40 late promoter in both cell culture and cell-free transcription systems. It was also shown to interact with both ER alpha and the transcription factor TFIIB by direct protein-protein contacts. Thus, hERR alpha 1 may play a role in the response of some genes to estrogen via heterodimerization with ERs or competition with ERs for binding to EREs.