Pdx-1 is a hox-like transcription factor that is responsible for the activation of insulin gene. Prior studies have demonstrated the interaction in vitro of Pdx-1 with short (20-40 nucleotide) DNA fragments corresponding to "A boxes" of the insulin promoter. Precisely how Pdx-1 binds to DNA in the complex milieu of chromatin, however, has never been studied. Here, we explored how Pdx-1-DNA interactions might be influenced by chromatin accessibility at the insulin gene in cells (TC3) vs. pancreatic ductal cells (mPAC). We demonstrate that Pdx-1 occupies the endogenous insulin promoter in TC3 cells but not in mPAC cells, a finding that is independent of the intracellular Pdx-1 protein concentration. Based on micrococcal nuclease protection assays, the difference in promoter binding between the two cell types appears to be secondary to chromatin accessibility at predicted Pdx-1 binding sites between bp -126 to -296 (relative to the transcriptional start site) of the insulin promoter. Binding studies using pruified Pdx-1 and reconsituted chromatin in vitro suggest that the positioning of a nucleosome(s) within this crucial region of the promoter might account for differences in chromatin accessibility. Consistent with these observations, fluorescence colocalization studies show that Pdx-1 does not occupy regions of compacted, nucleosome-rich chromatin within the nucleus. Our findings suggest a model whereby insulin transcription in the cell is at least partially facilitated by enahanced chromatin accessibility within a crucial regulatory region between bp -126 to -296, thereby permitting occupancy by transactivators such as Pdx-1.