The regulation mechanism of female-predominant expression of the mouse Cyp2b9 gene was investigated in vivo and in vitro. Luciferase reporter assay revealed that the -234/-194 region of the Cyp2b9 gene may be responsible for sexually dimorphic expression. There is a predicted forkhead box A2 (FoxA2) (hepatic nuclear factor 3beta)-binding site in this region. Chromatin immunoprecipitation assay indicated that the binding protein to the site was FoxA2 in 5-week-old female mice, whereas this protein was found in both sexes at age 3 weeks, in accordance with our previous observation on the developmental expression of this gene. Mutation of the predicted FoxA2 site in the reporter construct containing the -234/+18 fragment led to complete elimination of luciferase activity, but deletion of the -234/-194 region resulted in considerable transcriptional activity, suggesting that by mutating the FoxA2-binding site a potent suppressor might bind to eliminate activity, whereas by deleting this region it could not. Sexually dimorphic secretion of growth hormone is involved in female-predominant expression of the gene, and the -234/-194 region was also responsible for suppressing the expression by male-type secretion.