Fibroblast growth factors (FGFs) function as mitogens and morphogens during vertebrate development. In the present study, to characterise the regulatory mechanism of FGF8 gene expression in developing Xenopus embryos the upstream region of the Xenopus FGF8 (XFGF8) gene was isolated. The upstream region of the XFGF8 gene contains two putative binding sites for the SRY (sex-determining region Y)-box 2 (SOX2) transcription factor. A reporter assay with serially deleted constructs revealed that the putative SOX2-binding motif may be a critical cis-element for XFGF8 gene activation in developing Xenopus embryos. Furthermore, Xenopus SOX2 (XSOX2) physically interacted with the SOX2-binding motif within the upstream region of the XFGF8 gene in vitro and in vivo. Depletion of endogenous XSOX2 resulted in loss of XFGF8 gene expression in midbrain-hindbrain junction, auditory placode, lens placode and forebrain in developing Xenopus embryos. Collectively, our results suggest that XSOX2 directly upregulates XFGF8 gene expression in the early embryonic development of Xenopus