Sry in some varieties of Mus musculus domesticus fails to form normal testis when introduced into the C57BL/6J (B6) strain. We studied the developmental pattern of pre-Sertoli cells that express Sox9 by immunofluorescence and the profile levels of Sox9 transcripts by semiquantitative reverse transcriptase polymerase chain reaction and in situ hybridization in developing gonads of B6-Ytir mice. Sox9-positive cells (pre-Sertoli cells) appeared in all B6.Ytir genital ridges at 11.5 and 12.5 days postcoitum (dpc). However, at 13.5 dpc, Sox9-positive cells were not detected only in 50% of the B6.Ytir gonads compared with 100% of B6 gonads. Although pre-Sertoli cells formed the seminiferous cords after 14.5 dpc in the medial region of the B6.Ytir gonad, the cranial and caudal regions formed ovarian tissue. Further, B6.Ytir ovaries have lower levels of Sox9 than ovotestes at all fetal stages. These results suggest that although the pre-Sertoli cell lineage forms in B6.Ytir genital ridges, its further differentiation into Sertoli cells is apparently prevented. The cause may be the low levels of Sox9 and down-regulation of its product. Results suggest that inhibitory signals of Sox9 acting along the whole genital ridge or only at its cranial and/or caudal regions underlie formation of B6.Ytir ovaries or ovotestes, respectively. Furthermore, our results suggest that infertility of B6.Ytir females may be due to the abnormal presence of Sox9 transcripts in their ovaries.