Estrogen-related receptor alpha (ERR alpha) is an orphan member of the superfamily of nuclear hormone receptors. ERR alpha was initially isolated based on its sequence homology to the estrogen receptor but is not activated by classic estrogens. To identify possible physiologic functions for this orphan receptorwe cloned the mouse ERR alpha cDNA and used it to characterize the expression of ERR alpha transcripts and to identify potential ERR alpha target genes. RNA in situ hybridization studies detect ERR alpha transcripts in an organ-specific manner through mid- to late embryonic developmentwith persistent high-level expression in brown adipose tissue and intestinal mucosa. In the adult mouseERR alpha is most highly expressed in kidneyheartand brown adipocytestissues which preferentially metabolize fatty acids. Binding site selection experiments show that ERR alpha preferentially binds to an ERR alpha response element (ERRE) containing a single consensus half-siteTNAAGGTCA. An ERRE is present in the 5'-flanking region of the gene encoding medium-chain acyl coenzyme A dehydrogenase (MCAD)a key enzyme involved in the mitochondrial beta-oxidation of fat. The MCAD nuclear receptor response element 1 (NRRE-1) interacts in vitro with ERR alpha expressed in COS-7 cells. Supershift experiments show that endogenous ERR alpha present in nuclear extracts obtained from a brown fat tumor cell line (HIB) interacts with NRRE-1. In the absence of its putative ligandERR alpha does not activate the MCAD promoter in transient transfection studies; howevera VP16-ERR alpha chimera activates natural and synthetic promoters containing NRRE-1. In additionERR alpha efficiently represses retinoic acid induction mediated by NRRE-1. These results demonstrate that ERR alpha can control the expression of MCAD through the NRRE-1 and thus may play an important role in regulating cellular energy balance in vivo.