The pancreatic islet hormone glucagon stimulates hepatic glucose production and thus maintains blood glucose levels in the fasting state. Transcription factors of the Foxa [Fox (forkhead box) subclass A; also known as HNF-3 (hepatocyte nuclear factor-3)] family are required for cell-specific activation of the glucagon gene in pancreatic islet alpha-cells. However, their action on the glucagon gene is poorly understood. In the present study, comparative sequence analysis and molecular characterization using protein-DNA binding and transient transfection assays revealed that the well-characterized Foxa-binding site in the G2 enhancer element of the rat glucagon gene is not conserved in humans and that the human G2 sequence lacks basal enhancer activity. A novel Foxa site was identified that is conserved in rats, mice and humans. It mediates activation of the glucagon gene by Foxa proteins and confers cell-specific promoter activity in glucagon-producing pancreatic islet alpha-cell lines. In contrast with previously identified Foxa-binding sites in the glucagon promoter, which bind nuclear Foxa2, the novel Foxa site was found to bind preferentially Foxa1 in nuclear extracts of a glucagon-producing pancreatic islet alpha-cell line, offering a mechanism that explains the decrease in glucagon gene expression in Foxa1-deficient mice. This site is located just upstream of the TATA box (between -30 and -50), suggesting a role for Foxa proteins in addition to direct transcriptional activation, such as a role in opening the chromatin at the start site of transcription of the glucagon gene.