Inhibin is a dimeric peptide hormone produced in ovarian granulosa cells that suppresses FSH synthesis and secretion in the pituitary. Expression of inhibin alpha- and beta-subunit genes in the rodent ovary is positively regulated by FSH and negatively regulated after the preovulatory LH surge. We have investigated the role of the transcription factor CCAAT/enhancer-binding protein-beta (C/EBPbeta) in repressing the inhibin alpha-subunit gene. C/EBPbeta knockout mice fail to appropriately down-regulate inhibin alpha-subunit mRNA levels after treatment with human chorionic gonadotropin, indicating that C/EBPbeta may function to repress inhibin gene expression. The expression and regulation of C/EBPbeta were examined in rodent ovary, and these studies show that C/EBPbeta is expressed in ovary and granulosa cells and is induced in response to human chorionic gonadotropin. Transient cotransfections with an inhibin promoter-luciferase reporter in a mouse granulosa cell line, GRMO2 cells, show that C/EBPbeta is capable of repressing both basal and forskolin-stimulated inhibin gene promoter activities. An upstream binding site for C/EBPbeta in the inhibin alpha-subunit promoter was identified by electrophoretic mobility shift assays, which, when mutated, results in elevated inhibin promoter activity. However, C/EBPbeta also represses shorter promoter constructs lacking this site, and this component of repression is dependent on the more proximal promoter cAMP response element (CRE). Electrophoretic mobility shift assays show that C/EBPbeta effectively competes with CRE-binding protein for binding to this atypical CRE. Thus, there are two distinct mechanisms by which C/EBPbeta represses inhibin alpha-subunit gene expression in ovarian granulosa cells.