Japanese flounder (Paralichthys olivaceus), a teleost fish that has XX/ XY sex determination system, exhibits temperature-dependent sex determination. We have previously shown that high water temperature or an aromatase inhibitor treatment causes the sex-reversal from genetic females to phenotypic males and suppression of mRNA expression of ovary-type P450 aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens, in Japanese flounder. These results strongly suggest that suppression of the cyp19a1 mRNA expression and the resultant inhibition of estrogen biosynthesis trigger the sex-reversal of the genetic females by high water temperature. To elucidate the mechanism of transcriptional regulation of the cyp19a1 gene in Japanese flounder, we first examined mRNA expression of foxl2, which is a member of the winged helix/forkhead family of transcription factors and is known to be involved in ovarian development in some vertebrates. RT-PCR indicated that mRNA expression of foxl2 was detected highly in the ovary but not in the testis, similar to that of cyp19a1. Moreover, transient transfection assay showed that the flounder Foxl2 could activate the cyp19a1 gene transcription in vitro. Therefore, we suggest that Foxl2 up-regulates cyp19a1 gene expression in Japanese flounder.