Sox proteins are expressed at many stages of development and in numerous tissues. The transcription factor Sox-2 is first expressed throughout the inner cell mass and subsequently becomes localized to the primitive ectoderm, developing central nervous system, and the lens. Sox-2 is also highly expressed in F9 embryonal carcinoma cells, but becomes undetectable following differentiation of these cells. In this study, we have isolated, sequenced, and performed the first characterization of the Sox-2 promoter of any species. Approximately 2kb of the Sox-2 5'-flanking region has been sequenced and the primary transcription start site mapped by primer extension analysis. Additionally, two positive regulatory regions within the promoter region have been identified. We also show that expression of Sox-2 promoter/reporter gene constructs is reduced in differentiated EC cells as compared to their undifferentiated counterparts. Furthermore, we have identified a consensus inverted CCAAT box motif present in the Sox-2 promoter. Mutagenesis of this site significantly reduces the expression of Sox-2 promoter/reporter constructs. We also demonstrate that this CCAAT box motif can bind the trimeric transcription factor NF-Y.