Forkhead box protein L2 (FOXL2) is the earliest ovarian marker and plays an important role in the regulation of cholesterol and steroid metabolism, inflammation, apoptosis, and ovarian development and function. Mutations and deficiencies of the human FOXL2 gene have been shown to cause blepharophimosis-ptosis-epicanthus inversus syndrome as well as premature ovarian failure. Although Foxl2 interacts with steroidogenic factor 1 (Nr5a1) and up-regulates cyp19a1a gene transcription in fish, FOXL2 represses the transcriptional activity of the gene that codes for steroidogenic acute regulatory protein (Star) in mice. Most of the recent studies have heavily focused on the FOXL2 target genes (Star and Cyp19a1) in the ovaries. Hence, it is of importance to search for other downstream targets of FOXL2 and for the possibility of FOXL2 expression in nonovarian tissues. Herein, we demonstrate that the interplay between FOXL2 and NR5A1 regulates Star and melanocortin 2 receptor (Mc2r) gene expression in mammalian systems. Both FOXL2 and NR5A1 are expressed in ovarian and adrenal gland tissues. As expected, FOXL2 represses and NR5A1 enhances the promoter activity of Star. Notably, the promoter activity of Mc2r is activated by FOXL2 in a dose-dependent manner. Surprisingly, we found that FOXL2 and NR5A1 synergistically up-regulate the transcriptional activity of Mc2r. By mapping the Mc2r promoter, we provide evidence that distal NR5A1 response elements (-1410 and -975) are required for synergistic activation by FOXL2 and NR5A1. These results suggest that the interplay between FOXL2 and NR5A1 on the Mc2r promoter functions as a novel mechanism for regulating MC2R-mediated cell signaling as well as steroidogenesis in adrenal glands. © 2010 by the Society for the Study of Reproduction, Inc.