Upon FSH stimulation, many genes are acutely induced in granulosa cells. Gonadotropin-inducible ovarian transcription factor 1 (GIOT1) represents a novel member of the group of transcriptional repressors that belong to one such gene. To investigate the mechanism of this transcriptional activation, a rat GIOT1 promoter region was isolated and subsequently ligated to a luciferase vector and transfected to freshly prepared granulosa cells. A luciferase reporter gene driven by 0.8 kb of the GIOT1 5'-flanking region was highly expressed in response to FSH. Deletion and mutational analyses indicated that two response elements, including a steroidogenic factor 1 (SF-1) site and a cAMP response element (CRE), are required for the activation of the gene by FSH. Gel shift experiments also indicated that SF-1 and CRE binding protein specifically bind to each site. To reveal the relationship between SF-1 and the cAMP-dependent protein kinase A pathway, cotransfection was performed using SF-1-deficient cells. Although SF-1 and the catalytic subunit of protein kinase A individually caused a modest stimulation of the GIOT1 promoter, dramatic synergistic effects were observed in the case of cotransfection. Although the amount of SF-1 remained unchanged in response to FSH in granulosa cells, Dax-1 expression immediately decreased. The ectopic expression of Dax-1 inhibited the SF-1-dependent GIOT1 promoter activity. These results suggest that the synergistic action of CRE binding protein and SF-1 and the rapid down-regulation of Dax-1 are responsible for the acute induction of GIOT1 gene by gonadotropin.