Mutations in transcription factors hepatocyte nuclear factors (HNF)-1alpha and HNF-1beta cause maturity-onset diabetes of the young (MODY) types 3 and 5, respectively. HNF-1alpha and HNF-1beta mutations are well studied in some tissues, but the mechanism by which HNF-1alpha and HNF-1beta mutations affect sucrase-isomaltase (SI) transcription in the small intestine is unclear. We studied the effects of 13 HNF-1alpha mutants and 2 HNF-1beta mutants on human SI gene transcription, which were identified in subjects with MODY3 and MODY5, respectively. Transactivation activity of 11 HNF-1alpha and 2 HNF-1beta mutants was significantly lower than that of wild (wt)-HNF-1alpha and wt-HNF-1beta. Furthermore, in co-expression studies with mutant (mu)-HNF-1alpha/ wt-HNF-1beta and wt-HNF-1alpha/mu-HNF-1beta, the combination of mu-HNF-1alpha (P379fsdelCT and T539fsdelC)/wt-HNF-1beta impaired SI transcription, but the others were not remarkably different from wt-HNF-1alpha/wt-HNF-1beta. Although wt-HNF-1beta inhibited the transactivation activity of wt-HNF-1alpha on SI transcription, the inhibitory effect was reduced by 2 HNF-1beta mutants. These results suggest that SI transcription might tend to be unchanged or lower in MODY3, while occurring more in MODY5.