PDX-1 was shown to be expressed early during development in cells of both exocrine and endocrine origin; later it becomes restricted primarily to β-cells where it regulates the expression of β-cell-specific genes and mediates the glucose effect on insulin gene transcription. Therefore, it was important to identify the molecular mechanisms that specifically govern the expression of pdx-1 in the mature β-cell. To address this question, we analyzed 7 kb of the 5′ flanking region of the human pdx-1 gene. By transient transfections of β- and non-β-cell lines with different 5′ and 3′ deletions of that region, a strong β-cell-specific enhancer element located between -3.71 and -3.46 kb was revealed. We also sequenced about 4.5 kb of the human 5′ flanking region and compared it with that of the mouse pdx-1 gene. This comparison revealed three short conserved regions, designated PH1, PH2, and PH3. We showed that HNF-3β can bind and stimulate the activity of the human PH1 and PH2 elements in non-β-cells. Results reported by Wu et al. (7) and Sharma et al. (6) also indicate that expression of the mouse pdx-1 is controlled by an HNF-3-like element. Thus, it can be stated that at least some aspects of pdx-1 expression rely on the transcription factor HNF-3β. Because HNF-3β is not restricted to β-cells, the selective transcription of pdx-1 is likely to rely on additional factors. Our findings that the PH1 enhancer element binds both HNF-3β and PDX-1 and that mutations in each individual site dramatically impair its transcriptional activity suggest that these factors cooperate with one another. We therefore propose that a possible feedback mechanism might control the expression of pdx-1 at different stages during development.