Estrogen-related receptor α (ERRα) is an orphan member of the superfamily of nuclear hormone receptors. ERRα 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 receptor, we cloned the mouse ERRα cDNA and used it to characterize the expression of ERRα transcripts and to identify potential ERRα target genes. RNA in situ hybridization studies detect ERRα transcripts in an organ-specific manner through mid-to late embryonic development, with persistent high-level expression in brown adipose tissue and intestinal mucosa. In the adult mouse, ERRα is most highly expressed in kidney, heart, and brown adipocytes, tissues which preferentially metabolize fatty acids. Binding site selection experiments show that ERRα preferentially binds to an ERRα response element (ERRE) containing a single consensus half-site, TNAAGGTCA. 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 β-oxidation of fat. The MCAD nuclear receptor response element 1 (NRRE-1) interacts in vitro with ERRα expressed in COS-7 cells. Supershift experiments show that endogenous ERRα present in nuclear extracts obtained from a brown fat tumor cell line (HIB) interacts with NRRE-1. In the absence of its putative ligand, ERRα does not activate the MCAD promoter in transient transfection studies; however, a VP16-ERRα chimera activates natural and synthetic promoters containing NRRE-1. In addition, ERRα efficiently represses retinoic acid induction mediated by NRRE-1. These results demonstrate that ERRα can control the expression of MCAD through the NRRE-1 and thus may play an important role in regulating cellular energy balance in vivo.