Testis induction is associated with gonadal Sry and Sox9 expression in mammals. This study investigated whether Sry expression directly induces male-specific Sox9 activation during early phases of testis differentiation. We have established an XX sex-reversal mouse line carrying the Sry transgene driven by a weak basal promoter of the Hsp70.3 gene (Hsp-Sry), whereby the transgene was activated in the gonads along the entire anteroposterior axis from earlier stages. The effects of misexpression and overexpression of Sry on the spatiotemporal pattern of Sox9 expression were examined using both XX and XY gonads of Hsp-Sry transgenic embryos. It was shown that ectopic expression of Sry transcripts in the entire gonadal area from earlier stages promotes neither any advance in the timing nor any appreciable ectopic activation of endogenous Sox9 expression. Immediately after the onset of Sox9 activation, however, both the level of Sox9 expression and the number of SOX9-positive cells were significantly enhanced in Hsp-Sry/XY gonads, as compared with those in wild-type/XY and Hsp-Sry/XX gonads. These findings suggest that, although Sry is capable of up-regulating Sox9 expression dose-dependently, Sry mRNA expression alone is not likely to provide positional or timing information needed for male-specific Sox9 activation in developing XY gonads.