Mutations in the hepatocyte nuclear factor (HNF)-1alpha gene have been linked to subtype 3 of maturity-onset diabetes of the young (MODY), a disease characterized by a primary defect in insulin secretion. Here we show that the human GLUT2 gene is closely regulated by HNF-1alpha via sequences downstream of the transcriptional start site by interaction with transcriptional co-activator p300. The promoter region of the human GLUT2 gene was subcloned into luciferase expression plasmids that were transfected together with HNF-1alpha expression plasmid into a pancreatic beta-cell line, HIT-T15, to evaluate transcriptional activities. HNF-1alpha enhanced human GLUT2 promoter activity sixfold. Site-direct mutagenesis and footprint analyses showed that the HNF-1alpha binding site (+200 to +218) is critical in human GLUT2 gene expression. Furthermore, mammalian two-hybrid and immunoprecipitation studies revealed the transactivation domain of HNF-1alpha (amino acids 391-540) to interact with both the NH(2)-terminal region (amino acids 180-662) and the COOH-terminal region (amino acids 1,818-2,079) of p300. These findings demonstrated that HNF-1alpha binds to the 5'-untranslated region of GLUT2 and that p300 acts as a transcriptional co-activator for HNF-1alpha. In addition, these results provided new insight into the regulatory function of HNF-1alpha by suggesting a molecular basis for human GLUT2 gene expression.