Sry, a single-copy gene on the Y-chromosome, acts dominantly to trigger differentiation of a testis from a gonadal primordium that otherwise develops into an ovary in mammals. Sry encodes a protein with a DNA-binding domain and probably acts as a transcription factor. However, the mode of SRY action in testis determination remains largely unknown. In the present study, we detected the endogenous SRY protein in normal XY fetal mouse gonads by Western blotting and immunohistochemistry. The tissue-specificity and ontogeny of the detected protein were consistent with those of Sry transcripts. Immunofluorescent double labeling revealed that the SRY protein was detected in the Sertoli cell lineage and was swiftly down-regulated concurrently with testis cord organization. Surprisingly, however, the SRY protein was detected in the entire gonad from the onset of its expression, not in parallel to the spatiotemporal pattern of testis cord organization. The SRY protein was also detected in the entire region of all B6.YTIR fetal gonads, which were anticipated to undergo either partial or complete sex reversal. SRY down-regulation was considerably delayed, compared with control B6.XY gonads and was not associated with testis cord organization in B6.YTIR gonads. We conclude that the testis-determining pathway is impaired at the site of SRY action in the B6.YTIR gonad. Developmental Dynamics 233:612–622, 2005. © 2005 Wiley-Liss, Inc.