Cyp19 is expressed at a high level in the gonad of the female tadpole of the frog Rana rugosa during sex determination. To identify sequence elements important for expression of Cyp19, we isolated a genomic clone (approximately 40 kbp) carrying R. rugosa Cyp19 and analyzed the nucleotide sequence of the 5'-flanking region to search for potential transcription factor binding sites. Sox (SRY-related HMG box) protein and Sf1 binding sites were found in the ovary-specific promoter region of Cyp19. Because Sox3 is located on the sex chromosome in R. rugosa, we conducted the luciferase reporter assay in Xenopus A6 cells using the promoter region. Sox3 drove the reporter gene in the cells, but Sf1 did not. When sequential deletion of the 2.7 kbp Cyp19-promoter region was undertaken, a fragment spanning nucleotides -191 to +48 was sufficient to drive the transcription of the reporter gene. In site-directed mutagenesis, the binding site at -57 in the region was critical for Sox3 responsiveness. Sox3 lacking the HMG box had no ability to promote Cyp19 transcription. In addition, a chromatin immunoprecipitation (ChIP) assay showed that DNA fragments were enriched 8-fold, as determined by real-time PCR, when chromatin was immunoprecipitated with the anti-His antibody against His-tagged Sox3. The results, taken together, suggest that Sox3 activates Cyp19 transcription by its direct binding to the binding site of the Cyp19 promoter region. Sox3 appears to be a factor that directs indifferent gonads to develop into an ovary in R. rugosa.