Mutations in hepatocyte nuclear factor-1beta (HNF-1beta) lead to type 5 maturity-onset diabetes of the young (MODY5). Moreover, mutations in the HNF-1beta gene might cause multiorgan abnormalities including renal diseases, genital malformations, and abnormal liver function. The objective of this study was to investigate the molecular mechanism of diabetes mellitus, intrauterine growth retardation, and cholestasis observed in MODY5 patients. We analyzed the transactivity of wild-type and three mutant HNF-1beta on native human insulin, IGF-I, and multidrug resistance protein 2 (MRP2) promoters in combination with HNF-1alpha, using a reporter-assay system in transiently transfected mammalian cells. In the human insulin gene promoter, we found that the cooperation of HNF-1alpha and HNF-1beta is prominent. Absence of this cooperation was observed in all of the HNF-1beta mutants. In the human IGF-I and MRP2 promoters, we found that the HNF-1beta His153Asn (H153N) mutant had a mutant-specific repressive effect on both HNF-1alpha and wild-type HNF-1beta transactivity. Absence of the cooperation of HNF-1beta mutants with HNF-1alpha in the human insulin gene promoter might be one cause of defective insulin secretion. The H153N mutant-specific repression of HNF-1alpha and HNF-1beta transactivity in human IGF-I and MRP2 promoters might explain the case-specific clinical features of growth retardation and cholestasis observed only in early infancy. We found differential property of HNF-1alpha/HNF-1beta activity and the effect of HNF-1beta mutants by the promoters. We consider that analyses of HNF-1beta mutants on the intended human native promoters in combination with HNF-1alpha may be useful in investigating the molecular mechanisms of the various features in MODY5.