Sox3/SOX3 is a member of SOX gene family of transcription factors implicated in the control of diverse developmental processes. It has been shown that Sox3 is critical determinant of neurogenesis playing the role in specifying neural fate. Human SOX3 gene is transcriptionally up-regulated during retinoic acid (RA) induced differentiation of NT2/D1 embryonal carcinoma cell line, widely used in vitro model system for studying human neural differentiation. In order to define elements responsible for SOX3 transcriptional activation, series of deletion constructs of the 5' noncoding region of this gene have been tested. Based on these data, we have recently described one RA response element and now we report the presence of additional regulatory region, 31 bp in length, also involved in RA-induction of SOX3 gene. More precisely, functional analysis results have shown that this short region of SOX3 promoter is able to retain RA-inducibility. Also, retinoid receptor RXR alpha has been implicated in mediating SOX3 RA-dependent activation via 31 bp regulatory region. Further, direct interaction of recombinant RXR alpha protein with this region was confirmed by EMSA. Mutation analysis study, in which we compared RXR alpha binding affinity to wild type, versus various mutated 31 bp oligonucleotides, finally revealed the unique structure and sequence of this novel RA/RXR alpha response region.