Hypoglycaemia-inducing sulfonylureas, widely used in the treatment of non-insulin dependent diabetes mellitus (NIDDM), are known to stimulate insulin secretion by closing the ATP-sensitive K+ (KATP) channel. When these channels close, the cell membrane will depolarize, leading to an increased influx of Ca2+ and thereby initiating exocytosis of the insulin-containing secretory granules. Previous studies in rodent tissue have shown that, in addition to the effect on KATP channels, sulphonylureas have a direct stimulatory action on the exocytotic machinery. This 'distal' action occurs independently of the KATP channel effects. Here, we have investigated the molecular mechanisms of the exocytosis in single isolated human pancreaticβ-cells in control and diseased tissue. Isolated islets of Langerhans were obtained from 2 normoglycaemic donors and from 3 patients with the potentially lethal condition Persistent Hyperinsulinemic Hypoglycaemia of Infancy (PHHI). Genetic analysis revealed that two of the PHHI patients carried a familial mutation in exon 35 of the sulfonylurea receptor (SUR1) gene. SUR1 is one of the subunits forming the β-cell KATP channel. This mutation causes a severe truncation of the protein and loss of functional KATP channels. Despite lacking operational KATP channels none of the known mutations in the SUR1 gene was found in the third PHHI patient. Exocytosis of the secretory granules was measured in real-time in isolated cells using capacitance measurements. During the process of exocytosis, the membrance enclosing the secretory granules will be incorporated into the cell membrane leading to an increase in total cell surface area, a process that can be monitored as an increase in cell membrane capacitance in an individual cell. Infusion of 170nM Ca2+ through the recording pipette stimulated exocytosis inβ-cells isolated from both normal (n=12) and disease tissue (n=27). When tolbutamide (100μM) was added to the pipette solution a marked augmentation of secretion was observed in control β-cells (n=9), and in the non-familial PHHI β-cells lacking the severe truncation of SUR1 (n=9). By contrast, in β-cells obtained from patients with the familial form of the disease, tolbutamide prevented the Ca2+-dependent exocytosis (n=15). These data indicate that Ca2+-dependent exocytosis in human β-cells is augmented by sulphonylureas, and allude to the possibility that defects in the SUR1 gene may also be associated with altered regulation of secretion at a step(s) distal to the KATP channel in neonatal hyperinsulinism.