Members of the winged helix transcription factor family are known to regulate epithelial cell differentiation by regulating cell-specific gene expression. rWIN is a newly discovered member of the winged helix family shown to be present in the adult rat testis. In the testis the human homolog of rWIN, HFH-11, was localized to the germ cells (i.e. spermatocytes and spermatids) undergoing spermatogenesis. In the present study we show that rWIN is also expressed in testicular Sertoli cells. Sertoli cells are the epithelial component of the seminiferous tubule and provide both the cytoarchitectural support and the microenvironment for developing germ cells. The presence of rWIN in Sertoli cells was confirmed by Northern blot and RT-PCR analysis. The rWIN transcript size in the Sertoli cells was different from the germ cell transcript that is probably due to alternative splicing or modifications of the 3'-untranslated region. At least two spliced variants of rWIN were observed in the Sertoli cells corresponding to the deletion of an exon in the DNA-binding region. Long term stimulation of cultured Sertoli cells with the gonadotropin FSH down-regulated rWIN expression. In contrast, short-term stimulation (2 h) transiently up-regulated rWIN expression. The FSH-induced transient stimulation of rWIN precedes expression of the transferrin gene that is a marker of Sertoli cell differentiation. FSH-induced transferrin promoter activity was inhibited when cultured Sertoli cells were treated with an antisense oligonucleotide to rWIN. Interestingly, the constitutive overexpression of the DNA-binding domain of rWIN also down-regulated transferrin promoter activity. Analysis of the transferrin promoter with various deletion mutations suggested that rWIN acts at an upstream gene of the transferrin promoter. The results indicate that a transient up-regulation of rWIN in part mediates the ability of FSH to activate the transferrin promoter, which can be inhibited with a rWIN antisense oligonucleotide or constitutive expression of the rWIN DNA-binding domain. The current study demonstrates that rWIN acts as an early event gene for FSH actions on Sertoli cells and that rWIN appears to have a role in the regulation of Sertoli cell differentiated functions.