Previous studies have shown that transcription of the fibroblast growth factor-4 (FGF-4) gene by early embryonic cells is dependent upon a powerful distal enhancer located 3 kb downstream of the transcription start site within the untranslated region of the last exon. The transcription factors Sox-2 and Oct-3 cooperatively bind to critical cis-regulatory elements within the enhancer to synergistically activate transcription. Moreover, the co-activator p300 can mediate the synergistic activity of Sox-2 and Oct-3, and p300 associates with the FGF-4 enhancer in vivo. Embryonal carcinoma (EC) cells have been used extensively as a model system to study the regulation of the FGF-4 gene during early development. Recently, it has been suggested that suboptimal levels of Sox-2 expression in F9 EC cells limit the transcription of the FGF-4 gene. The studies presented in this report argue that Sox-2 levels are not limiting in F9 EC cells. Moreover, overexpression of Sox-2 in F9 EC cells decreases FGF-4 promoter activity. In addition, overexpression of Sox-2 in these cells inhibits activation by the co-activators p300, CBP, and OCA-B in a manner that requires the transactivation domain of Sox-2. These findings suggest that Sox-2 levels in F9 EC cells are regulated carefully to avoid interference with the transcription of critical genes.