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In B6.Ytir gonads that take the ovarian pathway, Sertoli cells expressing Sox9 protein disappeared beyond 13.5 dpc; we did detect Sox9 transcripts in B6.Ytir ovaries, but at significantly lower levels than in gonads that form seminiferous cords (at 17.5 dpc). Since Sox9 transcripts in B6.Ytir ovaries are lower than in B6 or CD1 testes, our results indicate that maintenance of Sox9 transcripts at threshold levels may be necessary for pre-Sertoli cells to undergo further differentiation. Although Sry can weakly initiate Sox9 expression in the B6.Ytir gonads at 12.5 dpc, its low activity or amount may lead to failure of Sox9-positive cells to further differentiate into Sertoli cells. Since Sertoli cells are necessary for testis tissue differentiation, it is tempting to speculate that lower levels of Sox9 transcripts in pre-Sertoli cells lead to B6.Ytir gonads developing as phenotypic ovaries. As in normal males, Sox9-positive cells appear in the genital ridges of B6.Ytir males in cells considered to be the pre-Sertoli cell lineage. Although this lineage appears in 100% of 11.5 and 12.5 dpc embryos, from 13.5 dpc onward the number of Sox9-positive cells diminishes and disappears in about half of B6.Ytir embryos. This observed loss of Sox9 protein suggests that unknown factors in B6.Ytir gonads prevent further differentiation of pre-Sertoli cells and lead them down the female pathway. The factors could be genetic modifiers that are manifested when Sry and/or Sox9 function is at threshold levels.