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Back crosses of M. musculus domesticus originated in Tirano, Italy (B6.Ytir), were used to study the time course expression of the Sox9 gene and its product in developing gonads. About half the embryos of each litter formed ovo-testis, whereas the other half formed ovaries as expected. Although in this strain the onset of Sry expression occurs normally, it lasts longer, suggesting that the male-determining cascade is modified in some way in the genital ridges of B6.Ytir embryos. We asked if failure to develop normal testes is at the level of Sox9, a gene that closely follows downstream of Sry expression and can substitute the role of Sry for complete testis differentiation. The polyclonal antibody used here was raised against the human SOX9 C-terminal 24 amino-acid epitope. This region forms part of the transcriptional activation domain and is highly conserved between species. The specificity of the antibody to Sox9 protein was previously shown to correlate with Sox9 mRNA by in situ hybridization in developing mice gonads. At 11.5 dpc, Sox9 was immunolocalized in the cytoplasm of several cells, suggesting that functional activity of Sox9 may depend on its nuclear translocation in pre-Sertoli cells. In the present study, however, Sox9 was always located within the nuclei of pre-Sertoli cells at 11.5 and 12.5 dpc, making it unlikely that the inhibition of differentiation of pre-Sertoli to Sertoli cells in B6.Ytir gonads is due to failure of nuclear translocation of Sox9.