The estrogen-related receptors (ERRalpha, -beta, and -gamma) are a subfamily of orphan nuclear receptors (designated NR3B1, NR3B2, and NR3B3) that are structurally and functionally related to estrogen receptors alpha and beta. Herein we test the hypothesis that ERRalpha regulates transcription of the genes encoding the enzymes involved in adrenal steroid production. Real-time RT-PCR was first used to determine the levels of ERRalpha mRNA in various human tissues. Adult adrenal levels of ERRalpha transcript were similar to that seen in heart, which is known to highly express ERRalpha. Expression of ERRalpha in the adult adrenal was then confirmed using Western blotting and immunohistochemistry. To examine the effects of ERRalpha on steroidogenic capacity we used reporter constructs with the 5'-flanking regions of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage (CYP11A), 3beta-hydroxysteroid dehydrogenase type II (HSD3B2), 17alpha-hydroxylase/17,20-lyase (CYP17), and dehydroepiandrosterone sulfotransferase (SULT2A1). Cotransfection of these reporter constructs with wild-type ERRalpha or VP16-ERRalpha expression vectors demonstrated ERRalpha enhanced reporter activity driven by flanking DNA from CYP17 and SULT2A1. SULT2A1 promoter activity was most responsive to the ERRalpha and VP16-ERRalpha, increasing activity 2.6- and 79.5-fold, respectively. ERRalpha effects on SULT2A1 were greater than the stimulation seen in response to steroidogenic factor 1 (SF1). Transfection of serial deletions of the 5'-flanking DNA of the SULT2A1 gene and EMSA experiments indicated the presence of three functional regulatory cis-elements which shared sequence similarity to binding sites for SF1. Taken together, the expression of ERRalpha in the adrenal and its regulation of SULT2A1 suggest an important role for this orphan receptor in the regulation of adrenal steroid production.