AbstractSry expression is essential for initiating male sex differentiation, and the expression occurs only during a restricted period in the developing gonad. It is thought that Sry is part of a pathway of genes that regulate sex determination. Although the interactions of several genes with Sry expression have been suggested, the exact cascade of gene expression regulating Sry transcription is entirely obscure because there is no available cell line expressing Sry and reflecting an in vivo condition. The present study was carried out to investigate the cis‐acting element of the mouse Sry that responds stage specifically to its expression, in part, using transgenic mice expressing GFP on the Y chromosome. Ten DNA fragments were generated by digesting the 5′ upstream region (positions 5491–8039; 2,549 bp) of mouse Sry with appropriate restriction enzymes. In an electrophoretic mobility assay with these fragments, the region from position 5491 to position 5799 (309 bp) was identified as forming specific protein–DNA complexes with nuclear extracts from 11.5 days post coitus (dpc) gonads, but not from 12.5 and 13.5‐dpc gonads. This region also formed specific protein–DNA complexes with the nuclear extracts from adult testicular germ cells that generate only a circular form from Sry. This stage‐specific responsive region was narrowed down to positions 5559–5616 by DNase I footprinting analysis. The assay of DNase I hypersensitive (HS) using the nuclear lysates from the 11.5‐dpc urogenital ridges demonstrated that the novel HS site was located in the proximity of position 5600. This region DNase I HS was also detected at the same position when the lysates from adult testicular germ cells were applied. The results indicate that the present HS site may be involved in the transcriptional regulation of the linear and/or circular molecule transcripts from mouse Sry gene. Mol. Reprod. Dev. 64: 389–396, 2003. © 2003 Wiley‐Liss, Inc.