We have previously described the cloning of the human reelin promoter and provided evidence that it is regulated, in part, through changes in methylation. Results from our current studies provide a more detailed analysis of this promoter and the interactions of the transcription factors Sp1 and paired box gene 6 (Pax6) with their recognition sites. The promoter was studied in NT2 cells which are a neuroprogenitor line that undergoes differentiation in vitro. We examined reelin mRNA and promoter induction following a 6-day treatment of these cells with retinoic acid (RA). Deletion and site-directed mutations showed functionally relevant sequences necessary for regulation. Gel-shift assays demonstrated that the main site of action of the promoter lies within a closely packed ( approximately 25 bp) region in which these transcription factors likely bind, possibly forming a DNA/protein complex. Based on our results, it appears likely that RA-induces reelin expression through a critical Sp1 site that resides adjacent to the Pax6 site within this multisite enhancer region. We show that induction of the reelin promoter with RA is accompanied by higher amounts of Sp1 and Pax6 binding to this region. Finally, we show that while mutations in the Sp1 site prevent the RA-mediated promoter induction, similar mutations in the Pax6 site do not. The data suggest that while the Pax6 site plays a role in modulating reelin expression, it is not absolutely required for induction by RA.