Occurrence of hypoglycemia during a prolonged fasting, accompanied by inappropriately high serum insulin levels, is considered a reliable index of the presence of insulinoma. Previous in vitro studies on insulin receptors, and in vivo hyperinsulinemic clamps have shown that patients with insulinoma are insulin resistant. In the present study 10 patients with insulinoma, 6 patients with nontumoral (also called functional or reactive hypoglycemia) hypoglycemia and 6 normal subjects were fasted for 24 h and their blood glucose levels were maintained constant by means of a programmed glucose infusion (isoglycemic glucose clamp) delivered by an artificial pancreas (Biostator). Blood glucose levels were monitored in continum, glucose infused (M) and glucose clearance (MCR) were evaluated at hourly intervals, and serum insulin (IRI) levels were evaluated every 6 h. Blood glucose levels were higher in controls than in patients with insulinoma and in patients with non tumoral hypoglycemia; M, MCR and IRI were progressively higher in controls, in patients with nontumoral hypoglycemia and in patients with insulinoma. The M/I index (M divided by IRI levels, an index of insulin sensitivity) was lower in patients with insulinoma than in other subjects and patients, indicating the existence of insulin resistance. These data indicate that: i) patients with insulinoma require large amounts of glucose to remain isoglycemic during a prolonged fast; ii) insulin resistance is a common feature of insulinoma and can be shown even under near physiologic conditions such as a 24-h fasting. The present study does not clarify whether insulin resistance occurs at the hepatic level or at other, peripheral levels.