Sox genes show diverse and dynamic patterns of expression and have been recognized as key players in the regulation of embryogenesis and nervous system development. SOX3 gene, an X-linked member of the family, is predominantly expressed in developing nervous system. Retinoic acid-induced differentiation of human teratocarcinoma NT2/D1 cells is marked by activation of SOX3 gene during early phases of induction. Previously, deletion analysis revealed that positive cis regulatory elements reside within -427 to -295 region of the human SOX3 promoter. Also, in vitro DNaseI footprinting assay showed that nuclear extracts from NT2/D1 cells bind to the sequence located 300 bp upstream from the transcription start site. Computer prediction software (MatInspector) was used to search the matrix family library database and to identify potential transcription binding sites within this protected region. This analysis revealed the presence of two adjacent binding sites for ZBP-89 transcription factor. Since this protein possesses multiple functions, including transcriptional regulation of a variety of genes and cell growth arrest, we sought to investigate its role in the transcriptional regulation of SOX3 gene. Gel mobility shift assays using NT2/D1 cell nucle ar extracts or recombinant GST protein, as well as supershift experiments, showed that ZBP-89 specifically binds to aforementioned protected region. Overexpression of ZBP-89 down-regulated SOX3 promoter-driven CAT activity both in uninduced and RA-induced NT2/D1 cells. Taken together, these results demonstrate that ZBP-89 functions as a repressor of basal and inducible expression of SOX3 gene.