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-Y^tir mice. Sox9-positive cells (pre-Sertoli cells) appeared in all B6.Y^tir 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.Y^tir 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.Y^tir gonad, the cranial and caudal regions formed ovarian tissue. Further, B6.Y^tir 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.Y^tir 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.Y^tir ovaries or ovotestes, respectively. Furthermore, our results suggest that infertility of B6.Y^tir females may be due to the abnormal presence of Sox9 transcripts in their ovaries.