Foxl2 is a transcription factor that plays a crucial role in the regulation of the early development of the female gonad in mammals and fish. However, little is known regarding its role in ovarian differentiation in amphibians. In this study, we isolated a Foxl2 cDNA from the ovary of the frog Rana rugosa and Xenopuslaevis and examined its expression during gonadal sex differentiation in R. rugosa. Alignment of known Foxl2 sequences from vertebrates showed high identity of the Foxl2 open reading frame and protein sequences, in particular the forkhead domain and C-terminal region, with other vertebrate sequences. Among different adult tissues, Foxl2 was expressed at its highest level in the ovary. Real-time RT-PCR analysis showed that Foxl2 expression was sexually dimorphic during gonadal sex differentiation in R. rugosa. In addition, Foxl2, which was detected immunochemically in somatic cells surrounding oocytes in the ovary, promoted R. rugosaCYP19 transcription in luciferase promoter assays conducted in A6 cells. We also found by FISH analysis that Foxl2 was an autosomal gene. Altogether, these results suggest that Foxl2 probably plays a very important role in ovarian differentiation of R. rugosa by possibly regulating CYP19 expression. The factor that up-regulates Foxl2 expression in female gonads still remains to be identified.