Maturity-onset diabetes of the young (MODY) is a monogenic, autosomal dominant subtype of early-onset diabetes mellitus due to defective insulin secretion by the pancreatic β-cell in humans. Five different genes have been identified including those encoding the tissue-specific transcription factors expressed in pancreatic β-cells, i.e. HNF-4α (MODY1), HNF-1α (MODY3), IPF-1 (also known as PDX-1, MODY4) and HNF-1β (MODY5). Analyzing the transcription of the HNF-4α gene, we now identify an alternative promoter, P2, which is 46 kb 5′ to the previously identified P1 promoter of the human gene. Based on RT–PCR this distant upstream P2 promoter represents the major transcription site in pancreatic β-cells, but is also used in hepatic cells. Transfection assays with various deletions and mutants of the P2 promoter reveal functional binding sites for HNF-1α, HNF-1β and IPF-1, the other transcription factors known to encode MODY genes. We demonstrate the significance of this alternative promoter in a large MODY family where a mutated IPF-1 binding site in the P2 promoter of the HNF-4α gene co-segregates with diabetes (LOD score 3.25). These data suggest a regulatory network of the four MODY transcription factors interconnected at the distant upstream P2 promoter of the HNF-4α gene.