Mutations in the beta-cell genes encoding the glycolytic enzyme glucokinase (GCK) and the transcription factor hepatocyte nuclear factor (HNF)-1alpha are the most common causes of maturity-onset diabetes of the young (MODY). Studying patients with mutations in these genes gives insights into the functions of these two critical beta-cell genes in humans. We studied 178 U.K. and French MODY family members, including 45 GCK mutation carriers and 40 HNF-1alpha mutation carriers. Homeostasis model assessment of fasting insulin and glucose showed reduced beta-cell function in both GCK (48% controls, P<0.0001) and HNF-1alpha (42% controls, P<0.0001). Insulin sensitivity was similar to that of control subjects in the GCK subjects (93% controls, P = 0.78) but increased in the HNF-1alpha subjects (134.5% controls, P = 0.005). The GCK patients showed a similar phenotype between and within families with mild lifelong fasting hyperglycemia (fasting plasma glucose [FPG] 5.5-9.2 mmol/l, interquartile [IQ] range 6.6-7.4), which declined slightly with age (0.017 mmol/l per year) and rarely required pharmacological treatment (17% oral hypoglycemic agents, 4% insulin). HNF-1alpha patients showed far greater variation in fasting glucose both between and within families (FPG 4.1-18.5 mmol/l, IQ range 5.45-10.4), with a marked deterioration with age (0.06 mmol/l per year), and 59% of patients required treatment with tablets or insulin. Proinsulin-to-insulin ratios are increased in HNF-1alpha subjects (29.5%) but not in GCK (18.5%) subjects. In an oral glucose tolerance test, the 0- to 120-min glucose increment was small in GCK patients (2.4+/-1.8 mmol/l) but large in HNF-1alpha patients (8.5+/-3.0 mmol/l, P< 0.0001). This comparison shows that the clear clinical differences in these two genetic subgroups of diabetes reflect the quantitative and qualitative differences in beta-cell dysfunction. The defect in GCK is a stable defect of glucose sensing, whereas the HNF-1alpha mutation causes a progressive defect that alters beta-cell insulin secretion directly rather than the sensing of glucose.