We investigated transacting factors binding to the cis-element important in tissue-specific expression of the human glucose transporter type 2 isoform (GLUT2) gene. By transient transfection assay, we determined that the 227-base pair fragment upstream of the ATG start site contained promoter activity and that the region from +87 to +132 (site C) was responsible for tissue-specific expression. DNase I footprinting and electrophoretic mobility shift assay indicated that site C contained one binding site for hepatocyte nuclear factor 1 (HNF1) and two binding sites for HNF3. The mutations at positions +101 and +103, which are considered to be critical in binding HNF1 and HNF3, resulted in a 53% decrease in promoter activity, whereas the mutation of the proximal HNF3 binding site (+115 and +117) reduced promoter activity by 28%. The mutations of these four sites resulted in marked decrease (70%) in promoter activity as well as diminished bindings of HNF1 and HNF3. A to G mutation, which causes conversion of the HNF1 and HNF3 binding sequence to the NF-Y binding site, resulted in a 22% decrease in promoter activity. We identified that both HNF1 and HNF3 function as transcriptional activators in GLUT2 gene expression. Coexpression of the pGL+74 (+74 to +301) construct with the HNF1alpha and HNF3beta expression vectors in NIH 3T3 cells showed the synergistic effect on GLUT2 promoter activity compared with the expression of HNF1alpha, HNF3beta, or a combination of HNF1beta and HNF3beta. These data suggest that HNF1alpha and HNF3beta may be the most important players in the tissue-specific expression of the human GLUT2 gene.